Compositions, methods and kits relating to REMODELIN

ABSTRACT

The invention relates to novel nucleic acids encoding a mammalian adventitia inducible and bone expressed gene designated REMODEL, and proteins encoded thereby, whose expression is increased in certain diseases, disorders, or conditions, including, but not limited to, negative remodeling, arterial restenosis, vessel injury, ectopic ossification, fibrosis, and the like. REMODELIN also plays a role in cell-cell and cell-matrix adhesion, bone density, bone formation, dorsal closure, bone mineralization, calcification/ossification, and is associated with spina bifida-like phenotype. In addition, the invention relates to affecting REMODELIN expression by administration of TGF-β and control of cellular gene expression using REMODELIN. The invention further relates to methods of treating and detecting these diseases, disorders or conditions, comprising modulating or detecting REMODELIN expression and/or production of REMODELIN polypeptide.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application is a continuation-in-part of U.S. patentapplication Ser. No. 09/692,081, filed on Oct. 19, 2000, from which itis entitled priority under 35 U.S.C. §120.

BACKGROUND OF THE INVENTION

[0002] The present invention relates to identifying novel processesinvolved in mediating arterial remodeling and formation of bone andcartilage.

[0003] Arterial stenosis with reduction in blood flow is a commonproblem in many vascular diseases. Several growth factors have beenimplicated in the mechanisms leading to vascular stenosis. For instance,fibroblast growth factor 2 (FGF-2) has been identified as an importantfactor in mediating proliferation of smooth muscle cells leading tointimal lesion formation. Furthermore, it has been demonstrated thatarterial stenosis in response to angioplasty is largely due to negativeremodeling as a result of adventitial fibrosis. As more fully set forthbelow, transforming growth factor beta (TGF-β) signaling has beendemonstrated to play an important role in arterial stenosis in that,among other things, inhibition of TGF-β signaling using a soluble TGF-βreceptor type II dramatically reduced lumen narrowing by decreasingnegative remodeling and adventitial matrix deposition as well bydecreasing neointima formation. These results indicate the crucial roleof TGF-β signaling in arterial response to injury.

[0004] Vascular remodeling is a response of blood vessels to bothphysiological and pathological stimuli, leading to either vesselenlargement (positive remodeling) or shrinkage (negative remodeling). Ithas been demonstrated that neointimal proliferation or intimal massfollowing angioplasty shows little correlation with restenosis becauseof permanent changes in vascular geometry (Kakuta et al., 1994,Circulation 89:2809-2815; Nunes et al., 1995, Arterioscler. Thromb.Vasc. Biol. 15:156-165). Negative remodeling has been shown to accountfor most of the restenosis process (Mintz et al., 1993, Circulation88:1-654), and is now generally considered the predominant cause ofrestenosis. A successful therapeutic approach to restenosis, therefore,would target negative vascular remodeling.

[0005] Several growth factors have been implicated in the mechanismsleading to vascular stenosis, such as fibroblast growth factor-2 (FGF-2)and transforming growth factor-β (TGF-β). Specifically, cellularresponses involving TGF-β in the adventitia have gained increasedattention for their potential involvement in adventitial remodeling(Wilcox et al., 1996, Int. J. Radiat. Oncol. Biol. Phys. 36:789-796;Wilcox and Scott, 1996, Int. J. Cardiol. 54S:S21-35; Shi et al., 1996,Circulation 93:340-348). There is evidence that proliferative eventsoccurring in the adventitia contribute to vascular remodeling andrestenosis in response to vascular injury (Wilcox et al., 1996, Int. J.Radiat. Oncol. Biol. Phys. 36:789-796; Wilcox et al., 1997, Ann. N.Y.Acad. Sci. 811:437-447; Scott et al., 1996, Circulation 93:2178-2187).There is now general agreement that TGF-β is a potential factor in theadventitial remodeling process (Shi et al., 1996, Arterioscler. Thromb.Vasc. Biol. 16:1298-1305).

[0006] Although it is known that the TGF-β family of cytokines can havea variety of effects on vascular cells, very little is known about therole of this family of cytokines in vascular remodeling. TGF-β affectsmany functions including proliferation of smooth muscle cells (SMC)(Halloran et al., 1995, Am. J. Surg. 170:193-197). It has beendemonstrated that inhibition of SMC proliferation by TGF-β1 occurs viaextension of the G2 phase of the cell cycle (Grainger et al., 1994,Biochem. J. 299:227-235). In contrast, it has also been shown thatinhibition of SMC proliferation by TGF-β1 is due to arrest in the lateG1 phase of the cell cycle (Reddy and Howe, 1993, J. Cell Physiol.156:48-55). SMC derived from atherosclerotic lesions responded to TGF-β1with an increase in proliferation, and lower levels of TGF-β receptor II(TGF-βRII) have been implicated in the lack of inhibition by TGF-β1 inthese cells (McCaffrey et al., 1995, J. Clin. Invest. 96:2667-2675).

[0007] Further studies have established that TGF-β1 stimulates SMCproliferation in vitro. Low doses of TGF-β1 stimulated SMC proliferationvia platelet-derived growth factor (PDGF)-amino acid (AA)-dependent andPDGF-AA-independent mechanisms, while higher doses of TGF-β1 wereinhibitory (Battegay et al., 1990, Cell 63:515-524; Stouffer and Owens,1994, J. Clin. Invest. 93:2048-2055). Bifunctional effects of TGF-β1 inmigration assays with SMC were also demonstrated (Koyama et al., 1990,Biochem. Biophys. Res. Commun. 169:725-729; Mii et al., 1993, Surgery114:464-470).

[0008] TGF-β1 also plays a role in intimal lesion formation as indicatedby a 5-7 fold induction of TGF-β1 mRNA in the balloon-injured ratcarotid artery, with elevated levels of TGF-β1 mRNA persisting for 2weeks (Majesky et al., 1991, J. Clin. Invest. 88:904-910). During the 2week period, elevated TGF-β1 mRNA levels correlated with increases inmRNA expression of fibronectin and alpha-2 (I) and alpha-1 (III)collagens. These studies also demonstrated that infusion of recombinantTGF-β1 caused an increase in intimal SMC proliferation in vivo (id.).

[0009] Among clinically significant findings regarding the role of TGF-βsignaling in arterial response to injury, it has been demonstrated thatTGF-β1 mRNA expression in restenotic lesions compared to primaryatherosclerotic lesions is increased (Nikol et al., 1992, J. Clin.Invest. 90:1582-1592). In the rat balloon injury model, treatment withTGF-β1 antibodies caused a small but significant reduction in neointimaformation (Wolf et al., 1994, J. Clin. Invest. 93:1172-1178).Overexpression of TGF-β1 in the rat carotid artery by adenoviral genetransfer led to transient neointima formation with cartilaginousmetaplasia that almost completely resolved within 8 weeks (Shulick etal., 1998, Proc. Natl. Acad. Sci. USA 95:6983-6988). Without wishing tobe bound by any particular theory, TGF-β1 may also effect vascular tonesince the factor was found to suppress nitric oxide synthase expression(Perella et al., 1996, J. Biol. Chem. 271:13776-13780) while at the sametime inducing the vasoconstrictor endothelin in SMC in vitro (Kuriharaet al., 1989, Biochem. Biophys. Res. Commun. 159:1435-1440). Further,TGF-β1 has been implicated in anti-apoptotic effects in SMC (Herbert andCarmeliet, 1997, FEBS Lett. 413:401-404).

[0010] Studies examining the expression of TGF-β ligand and TGF-βreceptor (TGF-βR) mRNAs using reverse transcriptase polymerase chainreaction (RT-PCR) analysis revealed that TGF-β1, TGF-β3, and TGF-βRIImRNA levels were increased in the media of the injured rat carotidartery (Ward et al., 1997, Arterioscler. Thromb. Vasc. Biol.17:2461-2470) and expression of TGF-β2 and TGF-β3 were also reported inSMC of the lung vasculature (Khalil et al., 1996, Am. J. Respir. CellMol. Biol. 14:131-138; Pelton et al., 1991, Am. J. Respir. Cell Mol.Biol. 5:522-530). However, reduced levels of TGF-βRII were demonstratedin human atherosclerotic lesions (McCaffrey et al., 1995, J. Clin.Invest. 96:2667-2675). The three TGF-β ligands have overlappingfunctions and all of them induce expression of the alpha-1 (I), alpha-2(I) and alpha-1 (III) chains of collagen (Bray et al., 1998,Hypertension 31:986-994).

[0011] The role of TGF-β isoforms in vascular repair processes wasexamined using a rat balloon catheter denudation model (Smith et al.,1999, Circ. Res. 84:1212-1222). Proliferating and quiescent SMC indenuded vessels expressed high levels of mRNA for TGF-β1, TGF-β2, andTGF-β3, and lower levels of TGF-βRII mRNA (Smith et al., 1999, Circ.Res. 84:1212-1222). The role of TGF-β signaling in the rat carotidartery balloon injury model was tested and it was shown that controlvessels developed an extensive neointima and adventitial fibrosis withabundant collagen production. Vessels from animals injected with arecombinant soluble TGF-βRII (designated as “TGF-βR:Fc”) revealed onlylittle neointima formation and much less collagen deposition in theadventitia. The adventitia also contained significantly fewer cells,indicating that the proliferation of adventitial fibroblasts is mediatedby TGF-β. Further, inhibition of TGF-β signaling with TGF-βR:Fcdramatically reduced lumen narrowing by decreasing negative remodelingand adventitial matrix deposition, as well as neointima formation.

[0012] TGF-β has been implicated in myofibroblastic transdifferentiation(Orlandi et al., 1994, Exp. Cell Res. 214:528-536; Desmouliere et al.,1993, J. Cell Biol. 122:103-1 1 1; Verbeek et al., 1994, Am. J. Pathol.144:372-382), causing fibroblasts to transiently express smooth muscleα-actin (Darby et al., 1990, Lab. Invest. 63:21-29). The expression ofsmooth muscle α-actin in the carotid artery was examined usingimmunostaining at 4 days after balloon denudation when proliferation ofadventitial fibroblasts is rapid. Immunoreactive smooth muscle α-actinwas either completely absent or markedly reduced in the outer adventitiaof vessels from rats treated with TGF-βR:Fc compared to controls. Thisresult demonstrated that the induction of smooth muscle α-actinexpression by adventitial fibroblasts is at least in part mediated byTGF-β.

[0013] Morphometric analysis of the carotid arteries demonstratedsignificant increases in lumen area in all rats treated with TGF-βR:Fcwith an approximate 88% increase with a dose of 2 mg/kg given everyother day for 2 weeks. Further, a dose of 0.5 mg/kg every other day for2 weeks caused nearly a 60% increase in lumen area despite the fact thatintimal lesion formation was not affected by this dose. These resultsindicate that loss of lumen area is in large part due to negativeremodeling and measurements of the perimeter of the neointima (IEL) andmedia (EEL) demonstrated that all doses of TGF-βR:Fc used in this studysignificantly inhibited the reduction in IEL and EEL.

[0014] The effect of TGF-βR:Fc on remodeling is highly relevant to theclinical situation of restenosis after angioplasty (Mintz et al., 1993,Circulation 88:1-654; Mintz et al., 1994, Circulation 90:1-24).Immunostaining with anti-human IgG antibody demonstrated that theTGF-βR:Fc primarily localized to the adventitia and neointima indicatingthat these are the predominant sites of TGF-β activity because TGF-βR:Fcbinds only active TGF-β. One prominent effect of soluble TGF-βRII wasthe effect on collagen synthesis, which was particularly striking in theadventitia of Masson's trichrome stained sections. It was further foundthat the effects of TGF-βR:Fc on collagen expression by Northern blotanalysis of RNA isolated from carotid arteries 4 days after injury weremarkedly reduced for collagen Type I and Type III, but Type XV wasunaffected. No differences in levels of osteopontin, tropoelastin, orfibronectin mRNA were detected.

[0015] Taken together, the aforementioned findings identify the TGF-βisoforms as major factors mediating adventitial fibrosis and negativeremodeling following vascular injury. Thus, genes whose expression isaffected by TGF-β are likely involved in such TGF-β associatedprocesses, including arterial stenosis mediated by, inter alia,adventitial fibrosis and negative remodeling.

[0016] Many of the factors and processes involved in artery repair areshared in the development of bone and cartilage. Bone remodeling is adynamic physiologic process by which bone mass is maintained or adjustedin response to appropriate stimuli (reviewed in Ducy et al., 2000,Science 289:1501-4.). This process consists of two phases, boneresorption by osteoclasts followed by bone deposition by osteoblasts.These events occur continually throughout the skeleton and understandingthe factors that regulate this process are likely to have importantimplications for the treatment of common bone diseases such asosteoporosis. Many vascular injury related genes have been found thatare known for their function in bone. These include osteopontin(Giachelli et al., 1993, J. Clin. Invest. 92:1686-1696.), alkalinephosphatase (ALP), bone morphogenic proteins (BMPs), and osteocalcin(Balica et al., 1997, Circulation 95:1954-60.; Bostrom et al., 1993, J.Clin. Invest. 91:1800-9.; Bostrom et al., 1995, Am. J. Cardiol.75:88B-91B). Calcifications frequently also occur dystrophically inarteries affected by atherosclerosis and it is a characteristic findingin the medial sclerosis of Mönckeberg. Furthermore, matrix Gla proteindeficient mice typically develop extensive vascular calcifications (Luoet al., 1997, Nature 386:78-81.). For a better understanding ofpotential common denominators between the events taking place in injuredarteries or tissues and bone/cartilage development, a brief descriptionof the cellular events is provided here.

[0017] Wound healing is characterized by the formation of granulationtissue from connective tissue surrounding the damaged area and itscomponents are inflammatory cells, proliferating fibroblasts andmyofibroblasts (smooth muscle α-actin positive), and a rich capillarynetwork. Matrix protein synthesis is abundant and as the wound closesand evolves into a scar, there is a decrease in cellularity associatedwith the disappearance of myofibroblasts (Desmouliere et al., 1995Am. J.Pathol. 146:56-66.). This cell loss occurs via apopototic cell death(Desmouliere et al., 1995Am. J. Pathol. 146:56-66.). A similar course ofevents can be observed in the adventitia of balloon-injured arterieswhere the response to injury leads to negative remodeling as thepredominant cause of restenosis. TGF-β is a major factor mediating theremodeling process in arteries in response to injury (Smith et al.,1999, Circ. Res. 84:1212-1222.). Inhibition of TGF-8 function with asoluble TGF-β receptor II completely blocks the transdifferentiation ofadventitial fibroblasts into myofibroblasts, demonstrating that thistransdifferentiation process is at least in part mediated by TGF-β(Smith et al., 1999, Circ. Res. 84:1212-1222.).

[0018] Chondrocytes participating in endochondral ossification undergoevents that have some resemblance with wound healing. These includeproliferation in the proliferative zone, followed by hypertrophy andsubsequent cell death by apoptosis. The ingrowth of blood vessels inthis process is critical as inhibition of vascular endothelial growthfactor-(VEGF) mediated angiogenesis causes a decrease in thehypertrophic zone with reduced bone formation (Gerber et al., 1999 Nat.Med. 5:623-8.). During this differentiation process matrix proteinsynthesis is abundant and TGF-B as well as BMP members have been shownto participate in it (Olsen et al., 2000, Annu. Rev. Cell Dev. Biol.16:191-220.).

[0019] An important gene for chondrocyte differentiation was firstrecognized through the discovery of a mutation that is associated with asevere dwarfing syndrome now referred to as campomelic dysplasia (Fosteret al., 1994, Nature 372:525-30.). This syndrome is characterized bymalformation of long bones, vertebrae, pelvic and skull bones. The geneaffected was Sox9, a member of the Sox gene family of transcriptionfactors. During mouse development Sox9 transcript expression peaks incartilage primordia at 11.5 to 14.5 dpc (Ng et al., 1997, Dev. Biol.183:108-21.) and like collagen II expression continues to be high inprechondrocytes and chondrocytes. In the growth plate of long bones,expression of the cartilage specific collagen II and Sox9 are seen inresting and proliferating chondrocytes. In hypertrophic chondrocytes,collagen II is still expressed while Sox9 is turned off. In the absenceof Sox9 no chondrocyte specific marker genes are expressed in Sox9 nullcells of mouse chimeras (Bi et al., 1999, Nat. Genet. 22:85-9.) and nodifferentiation of chondrocytes from the perichondrium takes place.While other Sox gene family members also participate in chondrocytespecific gene expression Sox9 appears to play a dominant role in thisprocess.

[0020] As part of the endochondral ossification process cartilage massincreases by proliferation of chondrocytes as well as deposition ofcartilage matrix. Once cartilage is formed chondrocytes located in thecentral region undergo further maturation into hypertrophicchondrocytes. These chondrocytes are characterized by their withdrawalfrom the cell cycle and the expression of collagen X which serves as amarker gene for these hypertrophic chondrocytes. With the recruitment ofblood vessels primary ossification centers are then formed. The matrixproduced by the hypertrophic chondrocytes is subsequently degraded andreplaced by trabecular bone synthesized by osteoblasts which arereplacing hypertrophic chondrocytes undergoing apoptosis.Simultaneously, a collar of compact bone is formed by osteoblastslocated in the perichondrium giving rise to the bone collar around theprimary ossification centers. At either end of the cartilage secondaryossification centers originate from the growth plate where a coordinatedprocess of chondrocyte proliferation, maturation, and apoptosis givesrise to longitudinal bone growth.

[0021] A member of the hedgehog family of genes, Indian hedgehog (Ihh),has been implicated as a key regulator of bone formation by promotingchondrocyte proliferation and inhibiting chondrocyte hypertrophy(Bitgood et al., 1995, Dev. Biol. 172:126-38.). The Ihh deficient mousehas a severe dwarfing syndrome due to the lack of endochondral boneformation (St-Jacques et al., 1999, Genes Dev. 13:2072-86.).Proliferation of chondrocytes is inhibited and chondrocyte maturation isdisturbed in these mice. Of interest is the fact that a constitutivelyactive receptor for the parathyroid hormone-related protein (PTHrP)under the control of the collagen IIalphal promoter can rescue thechondrocyte maturation phenotype of the Ihh deficient mouse. PTHrPsynthesis is stimulated by Ihh expressed by cells in the perichondrium(Vortkamp et al., 1996, Science 273:613-22.) and there appears to be afeedback mechanism between these two ligands regulating the relativeproportions of hypertrophic and proliferative chondrocytes. Conditionsassociated with both increased and decreased numbers of proliferativechondrocytes are associated with dwarfism as this results in decreasedformation of hypertrophic chondrocytes in both cases.

[0022] How expression of Ihh in the proliferating chondrocytes isregulated is currently not well understood. However, signaling via theFGFR3 (Chen et al., 1999, J. Clin. Invest. 104:1517-25.; Naski et al.,1998, Development 125:4977-88.) and the PTHrP receptor may inhibit whileBMPs may serve as inducers of Ihh expression (Pathi et al., 1999, Dev.Biol. 209:239-53.). It is of interest that Ihh deficiency has verylittle consequences for osteoblast differentiation occurring duringintramembraneous ossification (St-Jacques et al., 1999, Genes Dev.13:2072-86.).

[0023] The transcription factor Cbfa1 has been identified as a crucialfactor regulating osteoblast differentiation in both intramembraneousand endochondral ossification. In the absence of Cbfa1, mice develop nobone tissue although a normal cartilage skeleton is formed (Schiffrin,1995, Cardiology 86 Suppl. 1: 16-22.; Komori, et al., 199, Cell89:755-64.; Otto et al., 1997, Cell 89:765-71.). This demonstrates thatCbfa1 is not essential for chondrogenesis although in certain instanceschondrocyte hypertrophy appears to require Cbfa1. During embryonicdevelopment, Cbfal expression is restricted to cells destined todifferentiate either into chondrocytes or osteoblasts (Schiffrin, 1995,Cardiology 86 Suppl. 1:16-22.). Cbfa1 expression later becomesrestricted to osteoblasts with only low levels seen also in hypertrophicchondrocytes (Kim et al., 1999, Mech. Dev. 80:159-70.).

[0024] It is likely that other transcription factors act downstream ofCbfal since osteoblasts do not appear until 14.5 days post coitus (dpc)while Cbfal expression is detectable as early as 10.5 dpc in mouseembryos. Homeobox genes like Msx2, Hoxa-2 and Bapxl are likely upstreamof Cbfa1, regulating its expression (Tribioli et al., 1997, Mech. Dev.65:145-62.; Tribioli et al., 1999, Development 126:5699-711.; Satokataet al., 2000, Nat. Genet. 24:391-5.; Gendron-Maguire et al., 1993, Cell75:1317-31.; Kanzler et al., 1998, Development 125:2587-97.). Deficiencyin the homeobox gene Dlx5 in mice is associated with delayedintramembraneous ossification and to a lesser extent also withendochondral ossification (Simeone et al., 1994, Proc. Natl. Acad. Sci.USA 91:2250-4.). However, Cbfa1 expression is not affected by thisdeficiency, indicating that Dlx5 is either downstream of Cbfa1 or in adifferent signaling pathway. Downstream of Cbfa1 are several bone matrixproteins. Cbfa1 is essential for bone matrix production as several bonematrix proteins including collagen I, osteocalcin (an osteoblastspecific protein), bone sialoprotein, and alkaline phosphatase havebinding sites for Cbfa1 in their promoters (OSE2 binding site)(Schiffrin, 1995, Cardiology 86 Suppl. 1:16-22.). Several members of thebone morphogenic family (BMP) can induce Cbfa1 expression in vitro whileTGF-β inhibits the expression of Cbfa1 in osteoblasts in vitro andcontrols the levels of osteoblast differentiation in vivo (Schiffrin,1995, Cardiology 86 Suppl. 1: 16-22.).

[0025] The major structural components of cartilage and bone arecollagens I and II with essential roles in providing mechanicalstrength. Several disease entities are associated with mutations inthese types of collagen with phenotypes ranging from normal stature withosteoarthritis to lethality (Alliston et al., 2001, EMBO J 20:2254-72.;Mundlos et al., 1997, FASEB J. 11:227-33.). Collagens play importantroles in wound and fracture healing and consequently inhibited collagenformation will impair the healing process. Excessive collagen formationis also of clinical significance in the form of fibrosis in a variety oforgans and tissues. Among the many diseases associated with collagenmutations are osteogenesis imperfecta (OI; collagen I) with brittlebones, chondrodysplasias (collagen II), several subtypes ofEhlers-Danlos syndrome (EDS, collagen III and others), Alport syndrome(collagen IV) with nephritis, Bethlem myopathy (collagen VI), dystrophicepidermolysis bullosea (DEB; collagen VII) with separation of theepidermis from the dermis, and many others (for review (Spranger et al.,1994, Eur. J. Pediatr. 153:56-65.).

[0026] The common structural element in all collagens is a uniquetriple-helical conformation which can be identified by its repetitive(G-X-Y)_(n) amino acid sequence pattern. The conformation is stabilizedby glycine as every third residue. The most common mutations are singlebase substitutions that cause replacement of a single glycine (G) byanother amino acid. This results in interruption of the (G-X-Y)_(n)sequence pattern and defective folding occurs as a consequence (forreview see Myllyharju et al., 2001, Ann Med. 33:7-21). Collagensynthesis involves a number of post-translational modifications andfibril forming collagens are first synthesized as procollagen moleculeswith propeptide extensions at both the N- and C-terminal ends.

[0027] The intracellular steps involved in the assembly of procollagenmolecules from pro α-chains include cleavage of the signal peptide,hydroxylation of certain proline and lysine residues, glycosylation ofcertain asparagine residues, association of the C propetides, andformation of interchain and intrachain disulfide bonds. Followingassociation of the C propeptides a nucleus of the triple helix is formedin the C-terminal region and the triple helix is then propagated towardsthe N terminus in a zipper-like fashion. These C propeptides allow thetethering of the 3 chains thus promoting triple helix formation, aprocess that can also be performed by a transmembrane domain (Baum etal., 1999, Curr. Opin. Struct. Biol. 9:122-8). The hydroxylation ofproline residues in the Y position creates a unique region of 5-6consecutive G-X-hydroxyP(roline) triplets at the C terminus of thetriple-helix domain which are thought to act as a nucleation site forbringing the 3 chains into the correct dihedral angles and for formingthe correct interchain hydrogen bonds. Studies by Bulleid et al.(Bulleid et al., 1997, EMBO J. 16:6694-701) demonstrated that thishydroxyP rich region is required for nucleation but that 2 consecutiveG-X-hydroxyP triplets at the C terminus may be sufficient. During theirtransport from the endoplasmic reticulum (ER) through the Golgi stackslateral aggregation of the procollagen molecules and increasedcondensation of the aggregates occurs during this process resulting ingranule formation for secretion. In the extracellular space the N and Cpropeptides are cleaved and self-assembly of the collagen molecules intofibrils occurs followed by covalent cross-linking of collagen molecules(reviewed in Myllyhaiju et al., 2001, Ann. Med. 33:7-21).

[0028] Less abundant collagens in cartilage are collagens IX, X, and XIwhere collagens IX and XI have predominantly regulatory functions infibril assembly. Expression of collagen X is limited to hypertrophicchondrocytes in the endochondral ossification process (Alliston et al.,2001, EMBO J. 20:2254-72). Important cartilage proteoglycans includeperlecan and aggrecan. The latter forms large complexes with hyaluronicacid stabilized via link protein (Bulleid et al., 1997, EMBO J.16:6694-701). Mutations in both link protein as well as aggrecan areassociated with multiple skeletal abnormalities (Watanabe et al., 1998,J. Biochem, (Tokyo) 124:687-93.; Watanabe et al., 1194, Nat. Genet.7:154-7).

[0029] Effective treatment of bone/cartilage and vascular diseases isdependent on understanding the processes by which these systems develop.Arterial stenosis with reduction in blood flow is a common problem inmany vascular diseases and it is an important causal factor in themorbidity and mortality associated with these diseases. Despite the factthat various growth factors, especially TGF-β, have been implicated inarterial stenosis, very few factors involved in arterial stenosis havebeen identified and characterized. Nevertheless, the identification ofsuch factors is crucial in the development of diagnostics andtherapeutics for treatment of vascular diseases associated or mediatedby arterial stenosis. Thus, there is long-felt need for theidentification and characterization of factors associated with arterialstenosis. The present invention meets this need. Similarly, properformation of bone and cartilage is essential to normal development, andthe formation of collagen matrices is an integral part of this process.Mutations of collagen genes are among the most common resulting inskeletal abnormalities. While little is known about the factors thatmodulate collagen matrices, understanding this process is highlyrelevant to many human diseases involving virtually all organ systems.Thus, there is a long felt need for the identification andcharacterization of factors associated with bone formation. The presentinvention meets this need.

BRIEF SUMMARY OF THE INVENTION

[0030] The invention includes an isolated nucleic acid encoding amammalian REMODELIN, or a fragment thereof.

[0031] In one aspect, the nucleic acid shares at least about 33%sequence identity with a nucleic acid encoding at least one of ratREMODELIN (SEQ ID NO: 1), and a human REMODELIN (SEQ ID NO: 3).

[0032] The invention also includes an isolated nucleic acid encoding amammalian REMODELIN, wherein the amino acid sequence of the REMODELINshares at least about 6% sequence identity with an amino acid sequenceof at least one of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.

[0033] The invention further includes an isolated polypeptide comprisinga mammalian REMODELIN.

[0034] In one aspect, the mammalian REMODELIN molecule shares at leastabout 6% sequence identity with an amino acid sequence of at least oneof SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.

[0035] The invention includes an isolated nucleic acid encoding amammalian REMODELIN, or a fragment thereof, the nucleic acid furthercomprising a nucleic acid encoding a tag polypeptide covalently linkedthereto.

[0036] In one aspect, the tag polypeptide is selected from the groupconsisting of a green fluorescent protein tag polypeptide, an influenzavirus hemagglutinin tag polypeptide, a myc tag polypeptide, aglutathione-S-transferase tag polypeptide, a myc-pyruvate kinase tagpolypeptide, a His6 tag polypeptide, a FLAG tag polypeptide, and amaltose binding protein tag polypeptide.

[0037] The invention includes an isolated nucleic acid encoding amammalian REMODELIN, or a fragment thereof, the nucleic acid furthercomprising a nucleic acid specifying a promoter/regulatory sequenceoperably linked thereto.

[0038] The invention includes a vector comprising an isolated nucleicacid encoding a mammalian REMODELIN, or a fragment thereof.

[0039] In one aspect, the vector further comprises a nucleic acidspecifying a promoter/regulatory sequence operably linked thereto.

[0040] The invention includes a recombinant cell comprising an isolatednucleic acid encoding a mammalian REMODELIN, or a fragment thereof.

[0041] The invention includes a recombinant cell comprising a vectorcomprising an isolated nucleic acid encoding a mammalian REMODELIN, or afragment thereof.

[0042] The invention includes an isolated nucleic acid complementary tothe an isolated nucleic acid encoding a mammalian REMODELIN, or afragment thereof, the complementary nucleic acid being in an antisenseorientation. In one aspect, nucleic acid shares at least about 33%identity with a nucleic acid complementary with a nucleic acid havingthe sequence of at least one of a rat REMODELIN molecule (SEQ ID NO: 1),and a human REMODELIN molecule (SEQ ID NO: 3).

[0043] The invention includes a recombinant cell comprising an isolatednucleic acid complementary to the an isolated nucleic acid encoding amammalian REMODELIN, or a fragment thereof, the complementary nucleicacid being in an antisense orientation.

[0044] The invention includes an antibody that specifically binds with amammalian REMODELIN molecule polypeptide, or a fragment thereof

[0045] In one aspect, the antibody is selected from the group consistingof a polyclonal antibody, a monoclonal antibody, a humanized antibody, achimeric antibody, and a synthetic antibody.

[0046] The invention includes a composition comprising an antibody thatspecifically binds with a mammalian REMODELIN molecule polypeptide, or afragment thereof, and a pharmaceutically-acceptable carrier.

[0047] The invention includes a composition comprising an isolatednucleic acid complementary to the an isolated nucleic acid encoding amammalian REMODELIN, or a fragment thereof, the complementary nucleicacid being in an antisense orientation, and apharmaceutically-acceptable carrier.

[0048] The invention includes a composition comprising an isolatednucleic acid encoding a mammalian REMODELIN, or a fragment thereof, anda pharmaceutically-acceptable carrier.

[0049] The invention includes a composition comprising an isolatedpolypeptide comprising a mammalian REMODELIN and apharmaceutically-acceptable carrier.

[0050] The invention includes a transgenic non-human mammal comprisingan isolated nucleic acid encoding a mammalian REMODELIN, or a fragmentthereof.

[0051] The invention includes a method of treating a disease mediated byabnormal expression of a REMODELIN molecule in a human. The methodcomprises administering to a human patient afflicted with a diseasemediated by abnormal expression of a REMODELIN molecule a REMODELINmolecule expression-inhibiting amount of a composition comprising anisolated nucleic acid complementary to the an isolated nucleic acidencoding a mammalian REMODELIN, or a fragment thereof, the complementarynucleic acid being in an antisense orientation, and apharmaceutically-acceptable carrier.

[0052] In one aspect, the disease is selected from the group consistingof impaired wound healing, fibrosis of an organ, ectopic ossification,and hypertrophic scar formation.

[0053] The invention further includes a method of diagnosing arterialrestenosis in a mammal. The method comprises obtaining a biologicalsample from the mammal, assessing the level of REMODELIN in thebiological sample, and comparing the level of REMODELIN in thebiological sample with the level of REMODELIN in a biological sampleobtained from a like mammal not afflicted with arterial restenosis,wherein a higher level of REMODELIN in the biological sample from themammal compared with the level of REMODELIN in the biological samplefrom the like mammal is an indication that the mammal is afflicted witharterial restenosis, thereby diagnosing arterial restenosis in themammal.

[0054] In one aspect, the biological sample is selected from the groupconsisting of a blood vessel sample, and a damaged tissue sample.

[0055] The invention includes a method of diagnosing negative remodelingin a mammal. The method comprises obtaining a biological sample from themammal, assessing the level of REMODELIN in the biological sample, andcomparing the level of REMODELIN in the biological sample with the levelof REMODELIN in a biological sample obtained from a like mammal notafflicted with negative remodeling, wherein a higher level of REMODELINin the biological sample from the mammal compared with the level ofREMODELIN in the biological sample from the like mammal is an indicationthat the mammal is afflicted with negative remodeling, therebydiagnosing negative remodeling in the mammal.

[0056] The invention includes a method of diagnosing fibrosis in amammal. The method comprises obtaining a biological sample from themammal, assessing the level of REMODELIN in the biological sample, andcomparing the level of REMODELIN in the biological sample with the levelof REMODELIN in a biological sample obtained from a like mammal notafflicted with fibrosis, wherein a higher level of REMODELIN in thebiological sample from the mammal compared with the level of REMODELINin the biological sample from the like mammal is an indication that themammal is afflicted with fibrosis, thereby diagnosing fibrosis in themammal.

[0057] The invention includes a method of identifying a compound thataffects expression of REMODELIN in a cell. The method comprisescontacting a cell with a test compound and comparing the level ofREMODELIN expression in the cell with the level of REMODELIN expressionin an otherwise identical cell not contacted with the test compound,wherein a higher or lower level of REMODELIN expression in the cellcontacted with the test compound compared with the level of REMODELINexpression in the otherwise identical cell not contacted with the testcompound is an indication that the test compound affects expression ofREMODELIN in a cell. In one aspect, the invention includes a compoundidentified by this method.

[0058] The invention includes a method of identifying a compound thatreduces expression of REMODELIN in a cell. The method comprisescontacting a cell with a test compound and comparing the level ofREMODELIN expression in the cell with the level of REMODELIN expressionin an otherwise identical cell not contacted with the test compound,wherein a lower level of REMODELIN expression in the cell contacted withthe test compound compared with the level of REMODELIN expression in theotherwise identical cell not contacted with the test compound is anindication that the test compound reduces expression of REMODELIN in acell. In one aspect, the invention includes a compound identified bythis method.

[0059] The invention includes a method of identifying a compound thataffects TGF-β signaling. The method comprises contacting a cell with atest compound and comparing the level of REMODELIN expression in thecell with the level of REMODELIN expression in an otherwise identicalcell not contacted with the test compound, wherein a higher or lowerlevel of REMODELIN expression in the cell contacted with the testcompound compared with the level of REMODELIN expression in theotherwise identical cell not contacted with the test compound is anindication that the test compound affects TGF-β signaling in a cell.

[0060] The invention includes a kit for alleviating a disease mediatedby abnormal expression of a REMODELIN in a human. The kit comprises aREMODELIN expression-inhibiting amount of a composition comprising anisolated nucleic acid complementary to the an isolated nucleic acidencoding a mammalian REMODELIN, or a fragment thereof, the complementarynucleic acid being in an antisense orientation, and apharmaceutically-acceptable carrier, the kit further comprising anapplicator, and an instructional material for the use thereof.

[0061] In one aspect, the disease is selected from the group consistingof negative remodeling, arterial restenosis, vessel injury, fibrosis.

[0062] The invention includes a kit for alleviating a disease mediatedby abnormal expression of a REMODELIN in a human. The kit comprises aREMODELIN expression-inhibiting amount of an isolated nucleic acidencoding a mammalian REMODELIN, or a fragment thereof, and apharmaceutically-acceptable carrier. The kit further comprises anapplicator, and an instructional material for the use thereof.

[0063] The invention includes a kit for treating a bone disease in amammal. The kit comprises a REMODELIN expression-inhibiting amount of aninhibitor of REMODELIN expression. The kit further comprises anapplicator, and an instructional material for the use thereof.

[0064] The invention includes a kit for treating a cartilage disease ina mammal. The kit comprises a REMODELIN expression-inhibiting amount ofan inhibitor of REMODELIN expression, the kit further comprising anapplicator, and an instructional material for the use thereof.

[0065] The invention includes a kit for inhibiting tissue calcification.The kit comprises a REMODELIN expression-inhibiting amount of aninhibitor of REMODELIN expression, the kit further comprising anapplicator, and an instructional material for the use thereof.

[0066] In one aspect, the tissue calcification is calcification of atransplant.

[0067] In another aspect, the transplant is a heart valve transplant.

[0068] The invention includes a method of increasing REMODELINexpression in a mammal. The method comprises administering a REMODELINexpression increasing amount of TGF-β to the mammal, thereby increasingREMODELIN expression in the mammal.

[0069] The invention includes a method of reducing REMODELIN expressionin a mammal. The method comprises administering a REMODELIN expressionreducing amount of TGF-β receptor type II to the mammal, therebyinhibiting signaling via TGF-β receptor type II and reducing expressionof REMODELIN in the mammal.

[0070] The invention includes a method of affecting cellular geneexpression in a mammal. The method comprises administering a nucleicacid encoding REMODELIN to the mammal, thereby affecting cellular geneexpression in the mammal.

[0071] In one aspect, the cellular gene is selected from the groupconsisting of TGF-β1, collagen IIIαI, osteopontin, biglycan, alkalinephosphatase, and bone morphogenic protein 4.

[0072] In another aspect, the expression of osteopontin is dependent onCbfa1.

[0073] The invention includes a method of affecting cellular geneexpression in a mammal. The method comprises administering a nucleicacid antisense to a nucleic acid encoding REMODELIN to the mammal,thereby affecting cellular gene expression in the mammal.

[0074] The invention includes a method of treating bone disease in amammal in need of such treatment. The method comprises administering toa mammal afflicted with the bone disease a REMODELINexpression-inhibiting amount of an inhibitor of REMODELIN expression,thereby inhibiting REMODELIN expression and treating the bone disease inthe mammal.

[0075] In one aspect, the bone disease is osteogenesis imperfecta.

[0076] The invention includes a method of treating cartilage disease ina mammal in need of such treatment. The method comprises administeringto a mammal afflicted with the cartilage disease a REMODELINexpression-inhibiting amount of an inhibitor of REMODELIN expression,thereby inhibiting REMODELIN expression and treating the cartilagedisease in the mammal.

[0077] In one aspect, the collagen disease is selected from the groupconsisting of osteogenesis imperfecta (OI), dystrophic epidermolysisbullosea (DEB), and Bethlem myopathy.

[0078] The invention includes a method of diagnosing a bone disease in amammal. The method comprises obtaining a biological sample from themammal, assessing the level of REMODELIN in the biological sample, andcomparing the level of REMODELIN in the biological sample with the levelof REMODELIN in a biological sample obtained from an otherwise identicalmammal not afflicted with bone disease, wherein a higher level ofREMODELIN in the biological sample from the mammal compared with thelevel of REMODELIN in the biological sample from the like mammal is anindication that the mammal is afflicted with bone disease, therebydiagnosing the bone disease in the mammal.

[0079] In one aspect, the bone disease is osteogenesis imperfecta.

[0080] The invention includes a method of diagnosing a collagen diseasein a mammal. The method comprises obtaining a biological sample from themammal, assessing the level of REMODELIN in the biological sample, andcomparing the level of REMODELIN in the biological sample with the levelof REMODELIN in a biological sample obtained from an otherwise identicalmammal not afflicted with a collagen disease, wherein a higher level ofREMODELIN in the biological sample from the mammal compared with thelevel of REMODELIN in the biological sample from the like mammal is anindication that the mammal is afflicted with a collagen disease, therebydiagnosing the collagen disease in the mammal.

[0081] In one aspect, the collagen disease is selected from the groupconsisting of osteogenesis imperfecta (OI), dystrophic epidermolysisbullosea (DEB), and Bethlem myopathy.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWINGS

[0082] The foregoing summary, as well as the following detaileddescription of the invention, will be better understood when read inconjunction with the appended drawings. For the purpose of illustratingthe invention, there are shown in the drawings embodiment(s) which arepresently preferred. It should be understood, however, that invention isnot limited to the precise arrangements and instrumentalities shown. Inthe drawings:

[0083]FIG. 1A-1 is an image of a Northern blot depicting total RNAisolated from various rat organs probed with labeled REMODELIN(REMODELIN) cDNA. A significant 1.2 kb band was present in cultured rataortic smooth muscle cells, ballooned rat aorta, lung, and brain.Significantly lower REMODELIN mRNA levels were detected in other tissues(i.e., liver, thymus, spleen, kidney, heart, muscle, uterus, andtestis). A transcript of about 3.5 kb was detected in SMC upon longerexposure of the blot or loading of higher amount of RNA on the gel.

[0084]FIG. 1A-2 is an image depicting the gel used for Northern blotanalysis in FIG. 1A-1. The gel was stained with ethidium bromide.

[0085]FIG. 1B-1 is an image of a Northern blot depicting expression ofREMODELIN mRNA in 8 day balloon-injured rat carotid arteries and normalcarotid arteries. The data depicted herein demonstrate that REMODELINmRNA is expressed in the injured arteries only.

[0086]FIG. 1B-2 is an image depicting the gel used for Northern blotanalysis in FIG. 1B-1. The gel was stained with ethidium bromide.

[0087]FIG. 1C-1 is an image of a Northern blot depicting levels ofREMODELIN mRNA in MC3T3 cells. The data disclosed herein demonstratethat the levels of REMODELIN mRNA were increased by the addition of bonemorphogenetic protein-4 (BMP-4), with peak expression after 8 hours.

[0088]FIG. 1C-2 is an image of a Northern blot depicting levels ofREMODELIN mRNA in MC3T3 cells. The data disclosed herein demonstratethat the levels of REMODELIN mRNA were increased by the addition ofTGF-β, with peak expression after 8 hours.

[0089]FIG. 1C-3 is an image depicting the gel used for Northern blotanalysis in FIG. 1C-2. The gel was stained with ethidium bromide.

[0090]FIG. 2A is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (sequence, SEQ IDNO: 6). The arrowheads indicate the position of the internal elasticlamina. The image depicts normal carotid arteries and demonstrates nodetectable REMODELIN expression therein. The presence of silver grains,appearing as white specks under dark field illumination, indicatesREMODELIN expression.

[0091]FIG. 2B is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The arrowheads indicate the position of the internal elastic lamina. Theimage demonstrates strong REMODELIN expression limited to the adventitiaof 8 day balloon injured arteries. The presence of silver grains,appearing as white specks under dark field illumination, indicatesREMODELIN expression.

[0092]FIG. 2C is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The arrowheads indicate the position of the internal elastic lamina. Theimage demonstrates maintained but decreased REMODELIN expression in theadventitia two weeks post-injury. The presence of silver grains,appearing as white specks under dark field illumination, indicatesREMODELIN expression.

[0093]FIG. 2D is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The arrowheads indicate the position of the internal elastic lamina. Theimage demonstrates that at 4 weeks post-injury, expression levels ofREMODELIN expression were similar to levels detected in normal, controlvessels. The presence of silver grains, appearing as white specks underdark field illumination, indicates REMODELIN expression.

[0094]FIG. 2E is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The image depicts a transverse section of an 11.5 days post coitus (dpc)mouse embryo wherein REMODELIN expression is detectable in thedeveloping mesoderm. The presence of silver grains, appearing as whitespecks under dark field illumination, indicates REMODELIN expression.

[0095]FIG. 2F is an image depicting an in situ hybridization analysisusing [35S]-UTP labeled antisense REMODELIN riboprobe (sequence, SEQ IDNO: 6). The image depicts a 14.5 dpc mouse embryo expressing REMODELINin developing bone. The developing brain and bone are depicted. Theimage depicts that REMODELIN expression becomes limited to thedeveloping bone at later stages of embryo development. The presence ofsilver grains, appearing as white specks under dark field illumination,indicates REMODELIN expression.

[0096]FIG. 2G is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The image depicts REMODELIN expression in the bones of the snout in a14.5 dpc embryo. The presence of silver grains, appearing as whitespecks under dark field illumination, indicates REMODELIN expression.

[0097]FIG. 2H is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The image depicts strong detectable REMODELIN expression in the bone ofthe developing skull of a 14.5 dpc mouse embryo. The presence of silvergrains, appearing as white specks under dark field illumination,indicates REMODELIN expression.

[0098]FIG. 2I is an image depicting an in situ hybridization analysisusing[³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6). Theimage depicts osteoblasts adjacent to mineralized bone in a femur from arat pup depicting expression of REMODELIN mRNA. The arrowheads indicatethe transition from the osteoblast layer to the mineralized bone layerat the upper right portion of the image. The image demonstrates strongREMODELIN expression in osteoblasts along mineralized bone. The presenceof silver grains, appearing as white specks under dark fieldillumination, indicates REMODELIN expression.

[0099]FIG. 2J is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The image depicts osteoblasts adjacent to mineralized bone in a femurfrom a rat pup depicting expression of REMODELIN mRNA. The arrowheadsindicate the transition from the osteoblast layer to the mineralizedbone layer at the upper right portion of the image. The imagedemonstrates strong REMODELIN expression in osteoblasts alongmineralized bone. The presence of silver grains, appearing as whitespecks under dark field illumination, indicates REMODELIN expression.

[0100]FIG. 2K is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The image depicts expression of REMODELIN was undetectable in normalskin (the skin surface is located on the left side of the image). Thepresence of silver grains, appearing as white specks under dark fieldillumination, indicates REMODELIN expression.

[0101]FIG. 2L is an image depicting an in situ hybridization analysisusing [³⁵S]-UTP labeled antisense REMODELIN riboprobe (SEQ ID NO: 6).The image depicts extensive expression of REMODELIN in a 7 day old skinincision along the wound edge in (myo)fibroblasts of the granulationtissue. The presence of silver grains, appearing as white specks underdark field illumination, indicates REMODELIN expression.

[0102]FIG. 3 is a diagram depicting the putative domains within the ratREMODELIN-short (REMODELINs) protein. The following domains areindicated: transmembrane domain/signal peptide (amino acid residues fromabout 1 to 32); a CK2 phosphorylation domain (amino acid residues fromabout 31 to 34); an N-myristoylation domain (amino acid residues fromabout 69 to 74); a CK2 phosphorylation domain (amino acid residues fromabout 99 to 102); an N-myristoylation domain (amino acid residues fromabout 119 to 124); a PKC phosphorylation domain (amino acid residuesfrom about 146 to 148); an N-myristoylation domain (amino acid residuesfrom about 165 to 170); an N-glycosylation domain (amino acid residuesfrom about 188 to 191); a CK2 phosphorylation domain (amino acidresidues from about 197 to 200); an N-myristoylation domain (amino acidresidues from about 201 to 206); an N-myristoylation domain (amino acidresidues from about 205 to 210); and a CK2 phosphorylation domain (aminoacid residues from about 219 to 222).

[0103]FIG. 4A is an image depicting the nucleic acid sequences for therat (SEQ ID NO: 1) and human (SEQ ID NO: 3) REMODELIN cDNA. Sequencehomology between rat and human REMODELIN cDNA is about 78% at the aminoacid level. Translational start sites and stop codons are underlined.Gaps introduced into a sequence to maximize the alignment are indicatedby a dash (“−”).

[0104]FIG. 4B is an image depicting a comparison of the amino acidsequences of rat (SEQ ID NO: 2) and human (SEQ ID NO: 4) REMODELIN. Thedata disclosed demonstrate that the two proteins share about 95%sequence identity. A consensus sequence is depicted between the twosequences. The “+” indicates a conserved amino acid substitution whereas“−” indicates either a gap or non-conserved amino acid substitution.

[0105]FIG. 4C is an image depicting the amino acid sequence of the longform of rat REMODELIN (rREMODELIN_(L)) (SEQ ID NO: 5), encoded by theisolated nucleic acid SEQ ID NO: 1 depicted in FIG. 4A, supra.

[0106]FIG. 5A is an image of an autoradiograph depicting expression ofREMODELIN protein using a rabbit reticulocyte lysate expression system.The image depicts the proteins produced by in vitro translation usingthe long and short forms of REMODELIN cDNA. Using the long form of therat REMODELIN cDNA that contains an additional 5′ in frame AUG startcodon as a template, a predominant 34 kDa protein was expressed andlesser amounts of a 30 kDa protein was detected. Only the 30 kDa proteinwas produced when translation was performed using the short form ofREMODELIN cDNA.

[0107]FIG. 5B is an image depicting NIH3T3 cells transfected with amyc-tagged REMODELIN (myc-REMODELIN) expression construct. Themyc-REMODELIN fusion protein product was detected using anti-mycantibody using confocal microscopy. The image depicts thatimmunoreactivity was observed throughout the cytoplasm in apunctate/vesicular pattern. Nuclear counterstain was performed usingpropidium iodide.

[0108]FIG. 5C is an image of an immunoblot probed using rabbit antibodyraised by immunizing using the carboxyterminal 15 amino acid residues ofREMODELIN (i.e., anti-REMODELIN IgG). Cell lysates obtained from normalcarotid arteries, and 1, 4, 7, 14 and 28 day balloon injured rat carotidarteries were resolved using SDS-PAGE and the proteins were transferredby Western blotting. The REMODELIN antibody recognized a single band ofapproximately 34 kDa band only in the cell lysate prepared from theinjured vessel but not in the normal vessel (nor. carotid).

[0109]FIG. 5D is an image of an immunoblot probed using rabbitanti-REMODELIN IgG demonstrating expression of REMODELIN protein invarious cell lines from different species as follows: NIH3T3, bovineaortic epithelium (BAE), PAC-1 (a rat smooth muscle cell line), Ar75 (arat smooth muscle cell line), RASMC (rat aortic smooth muscle cells),293 cells, BASMC (bovine aortic SMC), 10T1/2 cells, human umbilical veinendothelial cells (HUVEC), A431 cells, and human aortic SMC (HASMC).

[0110]FIG. 5E is an image of an immunoblot probed using rabbitanti-REMODELIN IgG depicting the effect of TGF-β1 or soluble TGF-βreceptor type II (sol. TGF-βRII) on REMODELIN expression. MC3T3 cellswere treated with 1 ng/ml of TGF-β1 or 100 ng/ml TGF-βRII and the cellswere harvested at the times indicated in the image. The data discloseddemonstrate that TGF-β1 stimulated REMODELIN expression while TGF-βRIIinhibited REMODELIN expression. Approximately 30 micrograms of proteinwere loaded per lane.

[0111]FIG. 5F is an image depicting BAE cells transfected with amyc-tagged REMODELIN (myc-REMODELIN) expression construct. BAE weretransiently transfected with a myc-tagged REMODELIN expressionconstruct. Expression of the transfected REMODELIN fusion protein wasdetected using an anti-myc antibody and the data disclosed demonstratethat very little expression is detectable at 48 hours post-transfection.Without wishing to be bound by any particular theory, these data suggestloss of the transfected cells. BAE transfected with an unrelated protein(EP1) using the same vector as that used to prepare the myc-taggedREMODELIN construct demonstrated higher levels of fusion proteinexpression 48 hours after transfection.

[0112]FIG. 6 is a series of images of Northern blots hybridized with theindicated probes. RNA was isolated from NIH3T3 cells stably transfectedwith a REMODELIN expression vector and corresponding vector transfectedcells. The antisense REMODELIN transfected MC3T3 cells and correspondingvector transfected cells are the same as shown in FIG. 12. REMODELINoverexpressing cells showed reduced levels of TGF-β1, collagen III, andbiglycan while in antisense transfected cells, these levels wereelevated. Osteopontin and ALP were highly upregulated in the absence ofREMODELIN expression while BMP-4 was down-regulated. An ethidium bromidestained membrane is shown as a loading control.

[0113]FIG. 7 is a set of graphs depicting luciferase activity in NIH3T3cells transiently transfected with luciferase reporter constructs.Cbfa1-dependent luciferase activity was completely inhibited in thepresence of cotransfected remodelin. Luciferase activity under thecontrol of the osteopontin promoter was inhibited in the presence ofcotransfected remodelin.

[0114]FIG. 8 is an image of an immunoblot of recombinant remodelinprotein probed with an anti-His tag antibody.

[0115]FIG. 9A is an image depicting the resulting phenotypes in Xenopusembryos after injection of REMODELIN mRNA at the oocyte 2 cell stage. Atthe 17-cell stage, embryos injected with lacZ control RNA (shown on theleft side of the image) exhibited normal development while embryosinjected with REMODELIN mRNA (shown on the right side of the image)exhibited inhibition of neurectodermal cell migration.

[0116]FIG. 9B is an image depicting normal control embryos at the34-cell stage.

[0117]FIG. 9C is an image depicting REMODELIN-injected 34-cell stageembryos. The REMODELIN-injected embryos were smaller, distorted, anddemonstrated abnormal development of the head compared with controlembryos depicted in FIG. 6B.

[0118]FIG. 9D is an image depicting a REMODELIN-injected embryoexhibiting an unfused neurectoderm due to failure of the neural tissuecells to migrate.

[0119]FIG. 9E is an image depicting a REMODELIN-injected embryoexhibiting displaying the split tail phenotype common inREMODELIN-injected embryos.

[0120]FIG. 10 is an image depicting the nucleic acid sequence (SEQ IDNO: 9) of a myc-tagged REMODELIN construct.

[0121]FIG. 11 is an image of an immunoblot depicting remodelin proteinlevels in MC3T3-E1 cells stably transfected with control vector or fulllength rat antisense remodelin cDNA. Levels of remodelin protein inantisense transfectants were undetectable while levels were normal invector transfectants. The lower band is non-specific.

[0122]FIG. 12A is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith control vector and clonal populations were isolated. The datadisclosed demonstrate that vector-transfected cells were of acobblestone morphology. The image depicts a phase contrast image using200× original magnification.

[0123]FIG. 12B is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith control vector and clonal populations were isolated. The datadisclosed demonstrate that vector-transfected cells were of acobblestone morphology. The image depicts a phase contrast image using200× original magnification.

[0124]FIG. 12C is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith control vector and clonal populations were isolated. The datadisclosed demonstrate that vector-transfected cells were of acobblestone morphology. The image depicts a phase contrast image using200× original magnification.

[0125]FIG. 12D is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith full length rat antisense REMODELIN cDNA and clonal populationswere isolated. The data disclosed herein demonstrate that antisenseREMODELIN transfected cells exhibit a distinctly altered phenotype withless adhesion to the substratum and reduced cell-cell contacts comparedwith control vector transfected cells. The image further depicts theincreased number in dead cells and cell debris in the antisensetransfected cells. The image depicts a phase contrast image using 200×original magnification.

[0126]FIG. 12E is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith full length rat antisense REMODELIN cDNA and clonal populationswere isolated. The data disclosed herein demonstrate that antisenseREMODELIN transfected cells exhibit a distinctly altered phenotype withless adhesion to the substratum and reduced cell-cell contacts comparedwith control vector transfected cells. The image further depicts theincreased number in dead cells and cell debris in the antisensetransfected cells. The image depicts a phase contrast image using 200×original magnification.

[0127]FIG. 12F is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith full length rat antisense REMODELIN cDNA and clonal populationswere isolated. The data disclosed herein demonstrate that antisenseREMODELIN transfected cells exhibit a distinctly altered phenotype withless adhesion to the substratum and reduced cell-cell contacts comparedwith control vector transfected cells. The image further depicts theincreased number in dead cells and cell debris in the antisensetransfected cells. The image depicts a phase contrast image using 200×original magnification.

[0128]FIG. 12G is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith full length rat antisense REMODELIN cDNA and clonal populationswere isolated. The data disclosed herein demonstrate that antisenseREMODELIN transfected cells exhibit a distinctly altered phenotype withless adhesion to the substratum and reduced cell-cell contacts comparedwith control vector transfected cells. The image further depicts theincreased number in dead cells and cell debris in the antisensetransfected cells. The image depicts a phase contrast image using 200×original magnification.

[0129]FIG. 12H is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith full length rat antisense REMODELIN cDNA and clonal populationswere isolated. The data disclosed herein demonstrate that antisenseREMODELIN transfected cells exhibit a distinctly altered phenotype withless adhesion to the substratum and reduced cell-cell contacts comparedwith control vector transfected cells. The image further depicts theincreased number in dead cells and cell debris in the antisensetransfected cells. The image depicts a phase contrast image using 200×original magnification.

[0130]FIG. 12I is an image depicting the effect of REMODELIN on celladhesion and cell-cell contacts. MC3T3 cells were stably transfectedwith full length rat antisense REMODELIN cDNA and clonal populationswere isolated. The data disclosed herein demonstrate that antisenseREMODELIN transfected cells exhibit a distinctly altered phenotype withless adhesion to the substratum and reduced cell-cell contacts comparedwith control vector transfected cells. The image further depicts theincreased number in dead cells and cell debris in the antisensetransfected cells. The image depicts a phase contrast image using 200×original magnification.

[0131]FIG. 13A is a graph demonstrating that REMODELIN expression isassociated with and/or mediates increased cell turnover. MC3T3 cellswere transfected with control vector or full-length rat antisenseREMODELIN cDNA and clonal populations were isolated. Cells wereharvested at the time points indicated and cell numbers were determined.The data disclosed demonstrate that there was no increase in cell numberin the antisense transfected cells compared with control cells.

[0132]FIG. 13B is a graph demonstrating that REMODELIN expression isassociated with and/or mediates increased cell turnover. MC3T3 cellswere transfected with control vector or full-length rat antisenseREMODELIN cDNA and clonal populations were isolated. The cells werepulsed with [³H]-thymidine for 4 hours before measuring incorporation oftritium in DNA. The cells were harvested in parallel at the time pointsafter plating indicated and cell numbers were determined (FIG. 9A). Thedata disclosed demonstrate that there was increased cell turnover sincethere was increased [³H]-thymidine incorporation but there was noincrease in cell number in the antisense transfected cells compared withcontrol cells (FIG. 9A).

[0133]FIG. 14A is an image depicting one day old REMODELIN transgenicmouse pups. Transgenic mice expressing REMODELIN under the control ofthe cytomegalovirus (CMV) promoter/regulatory sequence were generatedand a transgenic female was bred with a transgenic male giving rise tothe pups depicted herein. All of the transgenic pups exhibitedhemorrhaging in the hip and shoulder regions.

[0134]FIG. 14B is an image depicting one day old REMODELIN transgenicmouse pups. Transgenic mice expressing REMODELIN under the control ofthe cytomegalovirus (CMV) promoter/regulatory sequence were generatedand a transgenic female was bred with a transgenic male giving rise tothe pups depicted herein. X-ray examination of the skeleton identifiedthat all transgenic mice were smaller with considerable shortening ofthe long bones. The image of three transgenic (“transg.”) and one normalmouse are depicted.

[0135]FIG. 14C is an image depicting one day old REMODELIN transgenicmouse pups. Transgenic mice expressing REMODELIN under the control ofthe cytomegalovirus (CMV) promoter/regulatory sequence were generatedand a transgenic female was bred with a transgenic male giving rise tothe pups depicted herein. Without wishing to be bound by any particulartheory, similar to a spina bifida phenotype, the transgenic miceexhibited protrusion of neural tissue through the dorsal muscle layersin the thoracic area.

[0136]FIG. 15A is an image depicting one day old transgenic mouse pupsexpressing REMODELIN under the control of the CMV promoter. Breeding ofa transgenic female with a transgenic male gave rise to the pupsdepicted. The arrowheads indicate hemorrhaging in the hip and shoulderregions.

[0137]FIG. 15B is an image depicting one day old transgenic mouse pupexpressing REMODELIN under the control of the CMV promoter. Breeding ofa transgenic female with a transgenic male gave rise to the pupdepicted. Skeletal preparations revealed fractures of the humerus.

[0138]FIG. 15C is an image depicting one day old transgenic mouse pupexpressing REMODELIN under the control of the CMV promoter. Breeding ofa transgenic female with a transgenic male gave rise to the pupdepicted. Skeletal preparations revealed fractures of the femur.

[0139]FIG. 15D is an image depicting an age matched normal mouse. Alcianblue staining of skeletal preparations is shown.

[0140]FIG. 15E is an image depicting one day old transgenic mouse pupexpressing REMODELIN under the control of the CMV promoter. Breeding ofa transgenic female with a transgenic male gave rise to the pupdepicted. Alcian blue staining was absent from the intervertebral jointsand the posterior portions of the vertebrae in REMODELIN transgenic micecompared to normal controls (see FIG. 15D).

[0141]FIG. 15F is an image depicting an age matched normal mouse.Cortical bone matrix of the tibia is shown.

[0142]FIG. 15G is an image depicting one day old transgenic mouse pupexpressing REMODELIN under the control of the CMV promoter. Breeding ofa transgenic female with a transgenic male gave rise to the pupdepicted. Cortical bone matrix of the tibia (stained blue; shown ingray) was a markedly reduced in transgenics compared to controls (seeFIG. 15F).

[0143]FIG. 15H is an image depicting a transgenic pup from atransgenic/wildtype cross with a more severe phenotype which diedprenatally. Severe skeletal abnormalities and separation of the skinwere evident.

[0144]FIG. 15I is an image depicting a transgenic pup from atransgenic/wildtype cross with a more severe phenotype which diedprenatally. X-ray analysis demonstrated dwarfism, a dramatic reductionin bone density, and malformations of all bones. (WT=wildtype;TG=REMODELIN transgenic).

[0145]FIG. 15J is an image depicting a transgenic pup from atransgenic/wildtype cross with a more severe phenotype which diedprenatally. Histologically, there was a considerable reduction in bonematrix (stained blue; shown in gray) in all bones including the skull.

[0146]FIG. 15K is an image depicting a transgenic pup from atransgenic/wildtype cross with a more severe phenotype which diedprenatally. Ribs had very little bone matrix.

[0147]FIG. 15L is an image depicting a transgenic pup from atransgenic/wildtype cross with a more severe phenotype which diedprenatally. Skin sections revealed separation of the epidermis from thedermis.

[0148]FIG. 16A is an image depicting skeletal preparation made from oneday old non-transgenic pups otherwise identical to REMODELIN transgenicpups. Mineralized bone appears pink in color (darker gray) and cartilageappears blue (lighter gray).

[0149]FIG. 16B is an image depicting skeletal preparation made from oneday old REMODELIN transgenic pups. Mineralized bone appears pink incolor (darker gray) and cartilage appears blue (lighter gray). The imagedepicts reduced cartilage formation in all bones, including the distalphalanges of the feet when compared with normal, non-transgenic pups(FIG. 11A).

[0150]FIG. 16C is an image depicting skeletal preparation made from oneday old non-transgenic pups otherwise identical to REMODELIN transgenicpups. Mineralized bone appears pink in color (darker gray) and cartilageappears blue (lighter gray).

[0151]FIG. 16D is an image depicting skeletal preparation made from oneday old REMODELIN transgenic pups. Mineralized bone appears pink incolor (darker gray) and cartilage appears blue (lighter gray). The imagedepicts that cartilage was absent from the intervertebral joints and theposterior portions of the vertebra when compared with normal,non-transgenic pups (FIG. 11C).

[0152]FIG. 16E is an image depicting skeletal preparation made from oneday old non-transgenic pups otherwise identical to REMODELIN transgenicpups. Mineralized bone appears pink in color (darker gray) and cartilageappears blue (lighter gray).

[0153]FIG. 16F is an image depicting skeletal preparation made from oneday old REMODELIN transgenic pups. Mineralized bone appears pink incolor (darker gray) and cartilage appears blue (lighter gray). The imagedepicts that the anterior portions of the ribs, particularly the morecaudal ones, exhibited a marked decrease in cartilage content whencompared with normal, non-transgenic pups (FIG. 11E).

[0154]FIG. 16G is an image depicting skeletal preparation made from oneday old non-transgenic pups otherwise identical to REMODELIN transgenicpups. Mineralized bone appears pink in color (darker gray) and cartilageappears blue (lighter gray).

[0155]FIG. 16H is an image depicting skeletal preparation made from oneday old REMODELIN transgenic pups. Mineralized bone appears pink incolor (darker gray) and cartilage appears blue (lighter gray). The imagedepicts that the transgenic pups exhibited decreased bone density,particularly in the flat bones of the skull which had a transparentappearance, when compared with normal, non-transgenic pups (FIG. 11G).

[0156]FIG. 16I is an image depicting skeletal preparation made from oneday old non-transgenic pups otherwise identical to REMODELIN transgenicpups. Mineralized bone appears pink in color (darker gray) and cartilageappears blue (lighter gray).

[0157]FIG. 16J is an image depicting skeletal preparation made from oneday old REMODELIN transgenic pups. Mineralized bone appears pink incolor (darker gray) and cartilage appears blue (lighter gray). The imagedepicts that the transgenic pups exhibited decreased bone densitycompared with normal, non-transgenic pups (FIG. 11I). The data discloseddemonstrate that decreased bone density is associated with fragilityleading to multiple fractures such as a fractured humerus (arrow), whichexplain the hemorrhaging observed in upper and lower limbs (FIG. 10A).

[0158]FIG. 17A is an image depicting endogenous REMODELIN expression innormal one day old mouse pups. In situ hybridization with antisenseREMODELIN RNA shows mRNA expression in the cartilage of the humerushead.

[0159]FIG. 17B is an image depicting endogenous REMODELIN expression innormal one day old mouse pups. Immunostaining with anti-REMODELIN IgG ona longitudinal section of the tibia.

[0160]FIG. 17C is an image depicting endogenous REMODELIN expression innormal one day old mouse pups. Higher magnification of a tibia headstained for REMODELIN.

[0161]FIG. 17D is an image depicting endogenous REMODELIN expression innormal one day old mouse pups. REMODELIN mRNA is expressed in periostealcells (PC) of the femur.

[0162]FIG. 17E is an image depicting endogenous REMODELIN expression innormal one day old mouse pups. Masson's Trichrome stained sectiondepicting cortical bone of the tibia.

[0163]FIG. 17F is an image depicting endogenous REMODELIN expression innormal one day old mouse pups. Similar section as shown in FIG. 17Edepicting some REMODELIN immunoreactivity in PC and osteoblasts (arrows)but not in osteocytes of the bone matrix (BM).

[0164]FIG. 17G is an image depicting expression of the REMODELIN-myctransgene in mice. Immunostaining with the anti-myc antibody wasperformed on a section of the arm depicting the proximal radius.

[0165]FIG. 17H is an image depicting expression of the REMODELIN-myctransgene in mice. High levels of the transgene proteins are expressedby osteocytes (arrowheads) and lower levels are expressed inosteoblasts.

[0166]FIG. 17I is an image depicting expression of the REMODELIN-myctransgene in mice. Preimmune IgG showed little background on section oftibia cartilage.

[0167]FIG. 18A is an image depicting one day old normal mice. EndogenousREMODELIN protein was not detectable in normal skin by immunostainingwith anti-REMODELIN IgG.

[0168]FIG. 18B is an image depicting one day old REMODELIN transgenicmice. High levels of the REMODELIN transgene were detected in theepidermis with anti-myc antibody and lower levels were detected in thedermis.

[0169]FIG. 18C is an image depicting one day old REMODELIN transgenicmice. The connective tissue of the dermis appeared very loose (Trichromestain).

[0170]FIG. 18D is an image depicting one day old normal mice. Skeletalmuscle in normal mice showed endogenous REMODELIN expression withanti-REMODELIN IgG immunostaining.

[0171]FIG. 18E is an image depicting cross-sectioned skeletal musclefibers from one day old normal mice.

[0172]FIG. 18F is an image depicting longitudinally-sectioned skeletalmuscle fibers from one day old normal mice.

[0173]FIG. 18G is an image depicting cross-sectioned skeletal musclefibers from one day old REMODELIN transgenic mice. The transgenespecific REMODELIN-myc antibody demonstrated high levels ofimmunoreactivity in skeletal muscle of transgenic mice and many of thefiber bundles were hollow (arrowheads).

[0174]FIG. 18H is an image depicting cross-sectioned skeletal musclefibers from one day old REMODELIN transgenic mice. Muscle fibers oftransgenic mice formed circular structures (arrowheads).

[0175]FIG. 18I is an image depicting cross-sectioned skeletal musclefibers from one day old REMODELIN transgenic mice. Muscle fibers oftransgenic mice were less densely packed.

[0176]FIG. 19 is an image depicting the sequence of an isolatedREMODELIN antisense ribonucleic acid (SEQ ID NO: 6) complementary to aportion of a nucleic acid encoding REMODELIN.

DETAILED DESCRIPTION OF THE INVENTION

[0177] The invention relates to the discovery of a novel nucleic acidencoding a mammalian adventitia-inducible bone expressed molecule termedREMODELIN, previously referred to as REMODEL and/or adventitia inducedbone expressed molecule (AIBE), and the proteins encoded thereby. Thedata disclosed herein demonstrate that REMODELIN plays a role in, interalia, arterial restenosis mediated by or associated with adventitialfibrosis. As described more fully below, REMODELIN also plays a role inbone and cartilage formation. Identification of REMODELIN has importantimplications in the development of therapeutics and diagnostics for,among other things, adventitial fibrosis, arterial restenosis, negativeremodeling, restenosis due to wound healing, and anti-cancer therapy.

[0178] More specifically, nucleic acids encoding REMODELIN have beenisolated in both rat and human. These sequences are provided herein, andhave no significant homology to any known cDNA sequence.

[0179] The data disclosed herein demonstrate that expression ofREMODELIN is induced by vessel injury in mammals. That is, REMODELIN wasexpressed in balloon-injured rat carotid arteries but not in normal,uninjured vessels. Furthermore, REMODELIN was expressed selectively inthe adventitia of the injured vessel, and was not expressed in theneointima or in the adventitia of normal vessels. Moreover, REMODELINexpression was induced by TGF-β. This is important since proliferativeevents occurring in the adventitia contribute to vascular remodeling andrestenosis in response to vascular injury and recent data demonstratethat TGF-β is a factor in this adventitial remodeling process. Thus,these data further indicate that REMODELIN plays a role in cellproliferation and/or migration associated with vessel injury andrestenosis due to negative remodeling.

[0180] The data disclosed herein also demonstrate that REMODELIN playsan important role in development of bone during mammalian embryogenesis.REMODELIN is normally expressed during mouse embryogenesis, butexpression is localized to developing bone. However, in the adult mouse,REMODELIN expression is virtually undetectable, expressing at very lowlevels in the adult brain and lung tissue.

[0181] Additionally, the data disclosed herein demonstrate that instudies using frog embryos, REMODELIN also plays a role in cellproliferation and/or migration in that expression of REMODELIN in frogembryos resulted in inhibition blastopore closure, failure of closure ofthe neural folds, formation of a split tail, and other developmentalabnormalities. The REMODELIN-injected embryos also presented withdecreased size and distortion and abnormal development of the head.

[0182] Injection of REMODELIN mRNA into frog embryos inhibitedFGF-induced mesoderm formation. That is, animal caps fromREMODELIN-injected embryos incubated with FGF-I resembled animal capsincubated in the absence of FGF-1. Indeed, the data disclosed hereindemonstrate that the phenotype observed in frog embryos injected withREMODELIN mRNA is similar to that of embryos injected from mRNA fordominant-negative FGF receptor constructs. These results, in addition tothe induction of REMODELIN by TGF-β, further indicate that REMODELIN isan important factor in cell proliferation, migration, or both.

[0183] Additionally, over-expression of REMODELIN in transgenic micegave rise to spina bifida-like spinal defects. The transgenic mouse pupsexhibited altered bone density and bone growth further indicating thatREMODELIN plays an important role in embryogenesis, including, but notlimited to, a role in bone growth and dorsal closure.

[0184] The data disclosed herein also demonstrate that REMODELIN islocalized in the cell membrane via five potential N-myristoylationsites. Without wishing to be bound by any particular theory, thesemyristoylation sites may serve to anchor REMODELIN protein in the cellmembrane. This would indicate that REMODELIN is not a secreted protein,but rather, it is associated with the cell in mediating its effect(s).

[0185] In sum, the data disclosed herein demonstrate that REMODELINplays a role in cell proliferation and/or migration and is involved incellular signaling. Furthermore, the data demonstrate that REMODELINlikely plays a role in adventitial fibrosis, negative remodeling andarterial restenosis, mediated by, among other things, smooth muscle cellproliferation. Therefore, the instant invention provides an in vitromodel for the study of the function and role(s) of REMODELIN in arterialremodeling, adventitial fibrosis, and restenosis in vessels, as well aspotential therapeutics and diagnostics for treatment of diseases,disorders or conditions associated with adventitial fibrosis, arterialrestenosis, bone density and bone growth.

[0186] Definitions

[0187] As used herein, each of the following terms has the meaningassociated with it in this section.

[0188] The articles “a” and “an” are used herein to refer to one or tomore than one (i.e., to at least one) of the grammatical object of thearticle. By way of example, “an element” means one element or more thanone element.

[0189] By the term “abnormal expression of REMODELIN,” as used herein,is meant that the level of expression of a REMODELIN molecule (e.g., ratREMODELIN_(S), rat REMODELIN_(L), human REMODELIN) in a cell isdetectably higher or lower than the level of expression of REMODELIN inan otherwise identical cell where the otherwise identical cell isobtained from, normal tissue that does not exhibit any detectabledisease, disorder or condition associated with or mediated by expressionof REMODELIN, such as, but not limited to, adventitial remodeling,adventitial fibrosis, arterial restenosis, negative remodeling, bonegrowth, bone fracture healing, wound healing in any tissue, and thelike.

[0190] As used herein, the term “adjacent” is used to refer tonucleotide sequences which are directly attached to one another, havingno intervening nucleotides. By way of example, the pentanucleotide5′-AAAAA-3′ is adjacent the trinucleotide 5′-TTT-3′ when the two areconnected thus: 5′-AAAAATTT-3′ or 5′-TTTAAAAA-3′, but not when the twoare connected thus: 5′-AAAAACTTT-3′.

[0191] As used herein, amino acids are represented by the full namethereof, by the three letter code corresponding thereto, or by theone-letter code corresponding thereto, as indicated in the followingtable: Full Name Three-Letter Code One-Letter Code Aspartic Acid Asp DGlutamic Acid Glu E Lysine Lys K Arginine Arg R Histidine His H TyrosineTyr Y Cysteine Cys C Asparagine Asn N Glutamine Gln Q Serine Ser SThreonine Thr T Glycine Gly G Alanine Ala A Valine Val V Leucine Leu LIsoleucine Ile I Methionine Met M Proline Pro P Phenylalanine Phe FTryptophan Trp W

[0192] By the term “adventitial fibrosis,” as used herein, is meant theextensive fibrous (connective) tissue formation in the outer layer(i.e., adventitia) of a blood vessel. Adventitial fibrosis is associatedwith abundant deposition of extracellular matrix and proliferation ofmyofibroblasts and fibroblasts.

[0193] As used herein, to “alleviate” a disease, disorder or conditionmeans reducing the severity of one or more symptoms of the disease,disorder or condition. This can include, but is not limited to, reducingthe level of REMODELIN expressed in a cell or tissue (e.g., smoothmuscle cell, lung tissue, an artery), reducing the level of cellproliferation and or migration, affecting wound healing, affectinggranulation tissue formation, affecting bone growth and/or fracturehealing, reducing negative remodeling, arterial restenosis and/oradventitial fibrosis, inhibiting premature calcification/ossification,inhibiting calcification of an implant (e.g. a heart valve), reducing orincreasing the level of REMODELIN in a patient, compared with the levelof REMODELIN in the patient prior to or in the absence of the method oftreatment, and the like.

[0194] “Antisense” refers particularly to the nucleic acid sequence ofthe non-coding strand of a double stranded DNA molecule encoding aprotein, or to a sequence which is substantially homologous to thenon-coding strand. As defined herein, an antisense sequence iscomplementary to the sequence of a double stranded DNA molecule encodinga protein. It is not necessary that the antisense sequence becomplementary solely to the coding portion of the coding strand of theDNA molecule. The antisense sequence may be complementary to regulatorysequences specified on the coding strand of a DNA molecule encoding aprotein, which regulatory sequences control expression of the codingsequences.

[0195] By the term “applicator” as the term is used herein, is meant anydevice including, but not limited to, a hypodermic syringe, a pipette, abronchoscope, a nebulizer, and the like, for administering the REMODELINnucleic acid, protein, and/or composition of the invention to a mammal.

[0196] “Arterial restenosis,” as that term is used herein, means there-narrowing of an artery in response to a vascular intervention aimedat dilating a stenosed (i.e., narrowed) artery.

[0197] “Biological sample,” as that term is used herein, means a sampleobtained from an animal that can be used to assess the level ofexpression of a REMODELIN, the level of REMODELIN protein present, orboth. Such a sample includes, but is not limited to, a blood vessel(e.g., carotid artery, aorta, and the like) sample, a lung tissuesample, a SMC sample, and a sample from any tissue undergoing woundhealing.

[0198] By “candidate anti-REMODELIN drug,” as the term is used herein,is meant a compound that when contacted with a cell, reduces the levelof expression of a nucleic acid encoding a REMODELIN protein in the cellcompared with the level of REMODELIN expression in that cell prior tocontacting the cell with the compound or which reduces the level ofexpression in the cell compared with the level of REMODELIN expressionin an otherwise identical cell which is not contacted with the compound.

[0199] A “cartilage disease,” is any disease, disorder or conditionassociated with, or mediated by, abnormal cartilage formation, modeling,and the like, compared to cartilage that is known to not be diseased.Typically, cartilage diseases are mediated by a mutation in a collagengene resulting in misfolding of the resultant mutant collagen proteincompared to the normal protein, thereby resulting in impaired function.

[0200] By “complementary to a portion or all of the nucleic acidencoding REMODELIN” is meant a sequence of nucleic acid which does notencode a REMODELIN protein. Rather, the sequence which is beingexpressed in the cells is identical to the non-coding strand of thenucleic acid encoding a REMODELIN protein and thus, does not encodeREMODELIN protein.

[0201] The terms “complementary” and “antisense” as used herein, are notentirely synonymous. “Antisense” refers particularly to the nucleic acidsequence of the non-coding strand of a double stranded DNA moleculeencoding a protein, or to a sequence which is substantially homologousto the non-coding strand.

[0202] “Complementary” as used herein refers to the broad concept ofsubunit sequence complementarity between two nucleic acids, e.g., twoDNA molecules. When a nucleotide position in both of the molecules isoccupied by nucleotides normally capable of base pairing with eachother, then the nucleic acids are considered to be complementary to eachother at this position. Thus, two nucleic acids are complementary toeach other when a substantial number (at least 50%) of correspondingpositions in each of the molecules are occupied by nucleotides whichnormally base pair with each other (e.g., A:T and G:C nucleotide pairs).As defined herein, an antisense sequence is complementary to thesequence of a double stranded DNA molecule encoding a protein. It is notnecessary that the antisense sequence be complementary solely to thecoding portion of the coding strand of the DNA molecule. The antisensesequence may be complementary to regulatory sequences specified on thecoding strand of a DNA molecule encoding a protein, which regulatorysequences control expression of the coding sequences.

[0203] A “coding region” of a gene consists of the nucleotide residuesof the coding strand of the gene and the nucleotides of the non-codingstrand of the gene which are homologous with or complementary to,respectively, the coding region of an mRNA molecule which is produced bytranscription of the gene.

[0204] A “coding region” of an mRNA molecule also consists of thenucleotide residues of the mRNA molecule which are matched with ananticodon region of a transfer RNA molecule during translation of themRNA molecule or which encode a stop codon. The coding region may thusinclude nucleotide residues corresponding to amino acid residues whichare not present in the mature protein encoded by the mRNA molecule(e.g., amino acid residues in a protein export signal sequence).

[0205] “Encoding” refers to the inherent property of specific sequencesof nucleotides in a polynucleotide, such as a gene, a cDNA, or an mRNA,to serve as templates for synthesis of other polymers and macromoleculesin biological processes having either a defined sequence of nucleotides(i.e., rRNA, tRNA and mRNA) or a defined sequence of amino acids and thebiological properties resulting therefrom. Thus, a gene encodes aprotein if transcription and translation of mRNA corresponding to thatgene produces the protein in a cell or other biological system. Both thecoding strand, the nucleotide sequence of which is identical to the mRNAsequence and is usually provided in sequence listings, and thenon-coding strand, used as the template for transcription of a gene orcDNA, can be referred to as encoding the protein or other product ofthat gene or cDNA.

[0206] Unless otherwise specified, a “nucleotide sequence encoding anamino acid sequence” includes all nucleotide sequences that aredegenerate versions of each other and that encode the same amino acidsequence. Nucleotide sequences that encode proteins and RNA may includeintrons.

[0207] “Expression vector” refers to a vector comprising a recombinantpolynucleotide comprising expression control sequences operativelylinked to a nucleotide sequence to be expressed. An expression vectorcomprises sufficient cis-acting elements for expression; other elementsfor expression can be supplied by the host cell or in an in vitroexpression system. Expression vectors include all those known in theart, such as cosmids, plasmids (e.g., naked or contained in liposomes)and viruses (e.g., retroviruses, adenoviruses, and adeno-associatedviruses) that incorporate the recombinant polynucleotide.

[0208] A first region of an oligonucleotide “flanks” a second region ofthe oligonucleotide if the two regions are adjacent one another or ifthe two regions are separated by no more than about 1000 nucleotideresidues, and preferably no more than about 100 nucleotide residues.

[0209] As used herein, the term “fragment” as applied to a nucleic acid,may ordinarily be at least about 20 nucleotides in length, preferably,at least about 50 nucleotides, more typically, from about 50 to about100 nucleotides, preferably, at least about 100 to about 200nucleotides, even more preferably, at least about 200 nucleotides toabout 300 nucleotides, yet even more preferably, at least about 300 toabout 350, even more preferably, at least about 350 nucleotides to about500 nucleotides, yet even more preferably, at least about 500 to about600, even more preferably, at least about 600 nucleotides to about 650nucleotides, yet even more preferably, at least about 650 to about 800,more preferably, from about 800 to about 1000 nucleotides, preferably,at least about 1000 to about 1100 nucleotides, even more preferably, atleast about 1100 nucleotides to about 1200 nucleotides, yet even morepreferably, at least about 1200 to about 1210, even more preferably, atleast about 1210 nucleotides to about 1220 nucleotides, yet even morepreferably, at least about 1220 to about 1225, and most preferably, thenucleic acid fragment will be greater than about 1230 nucleotides inlength.

[0210] However, as applied to a nucleic acid, the term fragment as usedherein does not encompass the following isolated nucleic acids asreferred to by their GenBank Accession numbers: AA335862 (sharing about87% identity over about 373 nucleotides with REMODELIN cDNA); C01758(sharing about 87% identity over about 356 nucleotides with REMODELINcDNA); AA335551 (sharing about 87% identity over about 334 nucleotideswith REMODELIN cDNA); AA406425 (sharing about 88% identity over about312 nucleotides with REMODELIN cDNA); R46857; AA584310; D79314;AI085616; D62262; AA482398; AA482544; AI359844; AI352209; AI239604;AI218433; AI081084; AI074870; AI074769; AA974239; AA969841; AA857920;AA723450; AA410434; AA738416; AI370649; AA507081.

[0211] As applied to a protein, a “fragment” of REMODELIN is about 20amino acids in length. More preferably, the fragment of a REMODELIN isabout 30 amino acids, even more preferably, at least about 40, yet morepreferably, at least about 60, even more preferably, at least about 80,yet more preferably, at least about 100, even more preferably, about100, and more preferably, at least about 150, more preferably, at leastabout 200, yet more preferably, at least about 240, even morepreferably, at least about 243, yet more preferably, at least about 250,even more preferably, about 270, and more preferably, at least about 277amino acids in length amino acids in length.

[0212] A “genomic DNA” is a DNA strand which has a nucleotide sequencehomologous with a gene. By way of example, both a fragment of achromosome and a cDNA derived by reverse transcription of a mammalianmRNA are genomic DNAs.

[0213] “Homologous” as used herein, refers to the subunit sequencesimilarity between two polymeric molecules, e.g., between two nucleicacid molecules, e.g., two DNA molecules or two RNA molecules, or betweentwo polypeptide molecules. When a subunit position in both of the twomolecules is occupied by the same monomeric subunit, e.g., if a positionin each of two DNA molecules is occupied by adenine, then they arehomologous at that position. The homology between two sequences is adirect function of the number of matching or homologous positions, e.g.,if half (e.g., five positions in a polymer ten subunits in length) ofthe positions in two compound sequences are homologous then the twosequences are 50% homologous, if 90% of the positions, e.g., 9 of 10,are matched or homologous, the two sequences share 90% homology. By wayof example, the DNA sequences 3′ATTGCC5′ and 3′TATGGC share 50%homology.

[0214] As used herein, “homology” is used synonymously with “identity.”

[0215] In addition, when the terms “homology” or “identity” are usedherein to refer to the nucleic acids and proteins, it should beconstrued to be applied to homology or identity at both the nucleic acidand the amino acid sequence levels.

[0216] A first oligonucleotide anneals with a second oligonucleotidewith “high stringency” or “under high stringency conditions” if the twooligonucleotides anneal under conditions whereby only oligonucleotideswhich are at least about 60%, more preferably at least about 65%, evenmore preferably at least about 70%, yet more preferably at least about80%, and preferably at least about 90% or, more preferably, at leastabout 95% complementary anneal with one another. The stringency ofconditions used to anneal two oligonucleotides is a function of, amongother factors, temperature, ionic strength of the annealing medium, theincubation period, the length of the oligonucleotides, the G-C contentof the oligonucleotides, and the expected degree of non-homology betweenthe two oligonucleotides, if known. Methods of adjusting the stringencyof annealing conditions are known (see, e.g., Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York).

[0217] The determination of percent identity between two nucleotide oramino acid sequences can be accomplished using a mathematical algorithm.For example, a mathematical algorithm useful for comparing two sequencesis the algorithm of Karlin and Altschul (1990, Proc. Natl. Acad. Sci.USA 87:2264-2268), modified as in Karlin and Altschul (1993, Proc. Natl.Acad. Sci. USA 90:5873-5877). This algorithm is incorporated into theNBLAST and XBLAST programs of Altschul et al. (1990, J. Mol. Biol.215:403-410), and can be accessed, for example, at the National Centerfor Biotechnology Information (NCBI) world wide web site having theuniversal resource locator “http://www.ncbi.nlm.nih.,sov/BLAST/”. BLASTnucleotide searches can be performed with the NBLAST program (designated“blastn” at the NCBI web site), using the following parameters: gappenalty=5; gap extension penalty=2; mismatch penalty=3; match reward=1;expectation value 10.0; and word size=11 to obtain nucleotide sequenceshomologous to a nucleic acid described herein. BLAST protein searchescan be performed with the XBLAST program (designated “blastn” at theNCBI web site) or the NCBI “blastp” program, using the followingparameters: expectation value 10.0, BLOSUM62 scoring matrix to obtainamino acid sequences homologous to a protein molecule described herein.

[0218] To obtain gapped alignments for comparison purposes, Gapped BLASTcan be utilized as described in Altschul et al. (1997, Nucleic AcidsRes. 25:3389-3402). Alternatively, PSI-Blast or PHI-Blast can be used toperform an iterated search which detects distant relationships betweenmolecules (id.) and relationships between molecules which share a commonpattern. When utilizing BLAST, Gapped BLAST, PSI-Blast, and PHI-Blastprograms, the default parameters of the respective programs (e.g.,XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0219] The percent identity between two sequences can be determinedusing techniques similar to those described above, with or withoutallowing gaps. In calculating percent identity, typically exact matchesare counted.

[0220] As used herein, the terms “gene” and “recombinant gene” refer tonucleic acid molecules comprising an open reading frame encoding apolypeptide of the invention. Such natural allelic variations cantypically result in 1-5% variance in the nucleotide sequence of a givengene. Alternative alleles can be identified by sequencing the gene ofinterest in a number of different individuals. This can be readilycarried out by using hybridization probes to identify the same geneticlocus in a variety of individuals. Any and all such nucleotidevariations and resulting amino acid polymorphisms or variations that arethe result of natural allelic variation and that do not alter thefunctional activity are intended to be within the scope of theinvention.

[0221] Moreover, nucleic acid molecules encoding proteins of theinvention from other species (homologs), which have a nucleotidesequence which differs from that of the mouse proteins described hereinare within the scope of the invention. Nucleic acid moleculescorresponding to natural allelic variants and homologs of a cDNA of theinvention can be isolated based on their identity to mouse nucleic acidmolecules using the mouse cDNAs, or a portion thereof, as ahybridization probe according to standard hybridization techniques understringent hybridization conditions. For example, a homolog of a nucleicacid encoding a rat REMODELIN protein of the invention can be isolatedbased on its hybridization with a nucleic acid molecule encoding all orpart of rat and/or human REMODELIN under high stringency conditions.

[0222] As used herein, an “instructional material” includes apublication, a recording, a diagram, or any other medium of expressionwhich can be used to communicate the usefulness of the nucleic acid,peptide, and/or composition of the invention in the kit for effectingalleviation of the various diseases or disorders recited herein.Optionally, or alternately, the instructional material may describe oneor more methods of alleviation the diseases or disorders in a cell or atissue of a mammal. The instructional material of the kit of theinvention may, for example, be affixed to a container which contains thenucleic acid, peptide, and/or composition of the invention or be shippedtogether with a container which contains the nucleic acid, peptide,and/or composition. Alternatively, the instructional material may beshipped separately from the container with the intention that theinstructional material and the compound be used cooperatively by therecipient.

[0223] An “isolated nucleic acid” refers to a nucleic acid segment orfragment which has been separated from sequences which flank it in anaturally occurring state, e.g., a DNA fragment which has been removedfrom the sequences which are normally adjacent to the fragment, e.g.,the sequences adjacent to the fragment in a genome in which it naturallyoccurs. The term also applies to nucleic acids which have beensubstantially purified from other components which naturally accompanythe nucleic acid, e.g., RNA or DNA or proteins, which naturallyaccompany it in the cell. The term therefore includes, for example, arecombinant DNA which is incorporated into a vector, into anautonomously replicating plasmid or virus, or into the genomic DNA of aprokaryote or eukaryote, or which exists as a separate molecule (e.g.,as a cDNA or a genomic or cDNA fragment produced by PCR or restrictionenzyme digestion) independent of other sequences. It also includes arecombinant DNA which is part of a hybrid gene encoding additionalpolypeptide sequence.

[0224] In the context of the present invention, the followingabbreviations for the commonly occurring nucleic acid bases are used.“A” refers to adenosine, “C” refers to cytidine, “G” refers toguanosine, “T” refers to thymidine, and “U” refers to uridine.

[0225] As used herein, the term “negative remodeling” (also known asinward remodeling) means a physiologic or pathologic response of a bloodvessel to a stimulus resulting in a reduction of vessel diameter andlumen diameter. Such a stimulus could be provided by, for example, butnot limited to, a change in blood flow or an angioplasty procedure.

[0226] “Neointima formation,” as that term is used herein, means thethickening and enlargement of the tunica intima of a blood vessel due toaccumulation of cells and extracellular matrix in this layer of thevessel.

[0227] By describing two polynucleotides as “operably linked” is meantthat a single-stranded or double-stranded nucleic acid moiety comprisesthe two polynucleotides arranged within the nucleic acid moiety in sucha manner that at least one of the two polynucleotides is able to exert aphysiological effect by which it is characterized upon the other. By wayof example, a promoter operably linked to the coding region of a gene isable to promote transcription of the coding region.

[0228] Preferably, when the nucleic acid encoding the desired proteinfurther comprises a promoter/regulatory sequence, thepromoter/regulatory is positioned at the 5′ end of the desired proteincoding sequence such that it drives expression of the desired protein ina cell. Together, the nucleic acid encoding the desired protein and itspromoter/regulatory sequence comprise a “transgene.”

[0229] As used herein, the term “promoter/regulatory sequence” means anucleic acid sequence which is required for expression of a gene productoperably linked to the promoter/regulatory sequence. In some instances,this sequence may be the core promoter sequence and in other instances,this sequence may also include an enhancer sequence and other regulatoryelements which are required for expression of the gene product. Thepromoter/regulatory sequence may, for example, be one which expressesthe gene product in a tissue specific manner.

[0230] A “constitutive” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellunder most or all physiological conditions of the cell.

[0231] An “inducible” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only when an inducer which corresponds to the promoter ispresent in the cell.

[0232] A “tissue-specific” promoter is a nucleotide sequence which, whenoperably linked with a polynucleotide which encodes or specifies a geneproduct, causes the gene product to be produced in a living human cellsubstantially only if the cell is a cell of the tissue typecorresponding to the promoter.

[0233] A “polyadenylation sequence” is a polynucleotide sequence whichdirects the addition of a poly A tail onto a transcribed messenger RNAsequence.

[0234] A “polynucleotide” means a single strand or parallel andanti-parallel strands of a nucleic acid. Thus, a polynucleotide may beeither a single-stranded or a double-stranded nucleic acid.

[0235] The term “nucleic acid” typically refers to largepolynucleotides.

[0236] The term “oligonucleotide” typically refers to shortpolynucleotides, generally, no greater than about 50 nucleotides. Itwill be understood that when a nucleotide sequence is represented by aDNA sequence (i.e., A, T, G, C), this also includes an RNA sequence(i.e., A, U, G, C) in which “U” replaces “T.”

[0237] Conventional notation is used herein to describe polynucleotidesequences: the left-hand end of a single-stranded polynucleotidesequence is the 5′-end; the left-hand direction of a double-strandedpolynucleotide sequence is referred to as the 5′-direction.

[0238] The direction of 5′ to 3′ addition of nucleotides to nascent RNAtranscripts is referred to as the transcription direction. The DNAstrand having the same sequence as an mRNA is referred to as the “codingstrand”; sequences on the DNA strand which are located 5′ to a referencepoint on the DNA are referred to as “upstream sequences”; sequences onthe DNA strand which are 3′ to a reference point on the DNA are referredto as “downstream sequences.”

[0239] A “portion” of a polynucleotide means at least at least abouttwenty sequential nucleotide residues of the polynucleotide. It isunderstood that a portion of a polynucleotide may include everynucleotide residue of the polynucleotide.

[0240] “Primer” refers to a polynucleotide that is capable ofspecifically hybridizing to a designated polynucleotide template andproviding a point of initiation for synthesis of a complementarypolynucleotide. Such synthesis occurs when the polynucleotide primer isplaced under conditions in which synthesis is induced, i.e., in thepresence of nucleotides, a complementary polynucleotide template, and anagent for polymerization such as DNA polymerase. A primer is typicallysingle-stranded, but may be double-stranded. Primers are typicallydeoxyribonucleic acids, but a wide variety of synthetic and naturallyoccurring primers are useful for many applications. A primer iscomplementary to the template to which it is designed to hybridize toserve as a site for the initiation of synthesis, but need not reflectthe exact sequence of the template. In such a case, specifichybridization of the primer to the template depends on the stringency ofthe hybridization conditions. Primers can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

[0241] “Probe” refers to a polynucleotide that is capable ofspecifically hybridizing to a designated sequence of anotherpolynucleotide. A probe specifically hybridizes to a targetcomplementary polynucleotide, but need not reflect the exactcomplementary sequence of the template. In such a case, specifichybridization of the probe to the target depends on the stringency ofthe hybridization conditions. Probes can be labeled with, e.g.,chromogenic, radioactive, or fluorescent moieties and used as detectablemoieties.

[0242] “Recombinant polynucleotide” refers to a polynucleotide havingsequences that are not naturally joined together. An amplified orassembled recombinant polynucleotide may be included in a suitablevector, and the vector can be used to transform a suitable host cell.

[0243] A recombinant polynucleotide may serve a non-coding function(e.g., promoter, origin of replication, ribosome-binding site, etc.) aswell.

[0244] A “recombinant polypeptide” is one which is produced uponexpression of a recombinant polynucleotide.

[0245] “Polypeptide” refers to a polymer composed of amino acidresidues, related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof linked via peptide bonds,related naturally occurring structural variants, and syntheticnon-naturally occurring analogs thereof. Synthetic polypeptides can besynthesized, for example, using an automated polypeptide synthesizer.

[0246] The term “protein” typically refers to large polypeptides.

[0247] The term “peptide” typically refers to short polypeptides.

[0248] Conventional notation is used herein to portray polypeptidesequences: the left-hand end of a polypeptide sequence is theamino-terminus; the right-hand end of a polypeptide sequence is thecarboxyl-terminus.

[0249] As used herein, the term “reporter gene” means a gene, theexpression of which can be detected using a known method. By way ofexample, the Escherichia coli lacZ gene may be used as a reporter genein a medium because expression of the lacZ gene can be detected usingknown methods by adding the chromogenic substrateo-nitrophenyl-β-galactoside to the medium (Gerhardt et al., eds., 1994,Methods for General and Molecular Bacteriology, American Society forMicrobiology, Washington, D.C., p. 574).

[0250] As used herein, the term “REMODELIN” means any adventitia-inducedand bone expressed molecule having significant sequence identity withREMODELIN disclosed herein. More specifically, the putative REMODELINwill share at least about 33% sequence identity with at least one of anucleic acid having the sequence SEQ ID NO: 1 and a nucleic acid havingthe sequence SEQ ID NO: 3. More preferably, the nucleic acid encodingREMODELIN has at least about 35% identity, even more preferably, atleast about 40% identity, yet more preferably, at least about 45%identity, even more preferably, at least about 50% identity, morepreferably, at least about 55% identity, even more preferably, at leastabout 60% identity, yet more preferably, at least about 65% identity,more preferably, at least about 70 % identity, yet more preferably, atleast about 75 % identity, even more preferably, at least about 80%identity, more preferably, at least about 85% identity, yet morepreferably, about 90% identity, even more preferably, at least about 95%identity, and most preferably, at least about 99% sequence identity withat least one of SEQ ID NO: 1 and SEQ ID NO: 3 disclosed herein. Evenmore preferably, the nucleic acid is at least one of SEQ ID NO: 1 andSEQ ID NO: 3. Further, the biological activity of a REMODELIN preferablyincludes inhibition of expression of the nucleic acid encoding theREMODELIN protein by a soluble TGF-β receptor type II (TGF-βRII), whichblocks TGF-β signaling. Further, preferably, the biological activity ofa REMODELIN molecule includes induction of expression of the nucleicacid by TGF, induction of expression of the nucleic acid encoding aREMODELIN in a blood vessel following vessel injury, induction ofexpression of the nucleic acid encoding the protein in fibroblastsduring wound healing, expression in osteoblasts during bone formation,causing cell death in endothelial cells when it is overexpressed,involvement in cell-cell and cell-matrix interaction, and affecting cellviability such as by, for example, affecting the life span of a cell.

[0251] Further, the data disclosed elsewhere herein demonstrate thatREMODELIN plays an important role in bone growth. In one embodiment,transgenic mice over-expressing REMODELIN, similar to data obtainedusing frog embryos which exhibited failure of dorsal closure, exhibitedspina bifida-like effects. Therefore, the term “REMODELIN” encompasses anucleic acid that, when over-expressed in a mammalian embryo, mediatesor is associated with altered bone growth, bone density, and/or spinabifida-like phenotype.

[0252] Unless otherwise indicated, “REMODELIN” encompasses all knownREMODELINs (e.g., rat REMODELIN_(S), rat REMODELIN_(L), and humanREMODELIN), and REMODELINs to be discovered, including but not limitedto, mouse REMODELIN, having the characteristics and/or physical featuresof the REMODELIN disclosed herein.

[0253] However, the present invention does not include the isolatednucleic acids having the sequences designated by the following GenBankAccession Numbers: AA335862 (sharing about 87% identity with REMODELINover about 373 nucleotides); C01758 (sharing about 87% identity withREMODELIN over about 356 nucleotides); AA335551 (sharing about 87%identity with REMODELIN over about 334 nucleotides); and AA406425(sharing about 88% identity with REMODELIN over about 312 nucleotides);R46857; AA584310; D79314; AI085616; D62262; AA482398; AA482544;AI359844; AI352209; AI239604; AI218433; AI081084; AI074870; AI074769;AA974239; AA969841; AA857920; AA723450; AA410434; AA738416; AI370649;AA507081.

[0254] “REMODELIN expression-inhibiting amount,” as used herein, meansany amount of a substance or molecule that detectably decreases thelevel of REMODELIN expression, amount, and/or activity compared with thelevel of REMODELIN expression, amount, and/or activity in the absence ofthe substance or molecule. Thus, any amount that mediates a detectabledecrease in: the amount of REMODELIN present, the level of REMODELINmRNA expression, and/or the ability of REMODELIN to form necessaryligand/receptor interactions, is encompassed in the present invention.The assays by which these conditions are examined are well-known in theart and several are exemplified herein.

[0255] “REMODELIN expression-increasing amount,” as used herein, meansany amount of a substance or molecule that detectably increases thelevel of REMODELIN expression, amount, and/or activity compared with thelevel of REMODELIN expression, amount, and/or activity in the absence ofthe substance or molecule. Thus, any amount that mediates a detectableincrease in: the amount of REMODELIN present, the level of REMODELINmRNA expression, and/or the ability of REMODELIN to form necessaryligand/receptor interactions, is encompassed in the present invention.The assays by which these conditions are examined are well-known in theart and several are exemplified herein.

[0256] By the term “REMODELIN-like activity,” as used herein, refers tothe ability of a molecule or compound to be induced by TGF-β,selectively induced in adventitia of injured vessels, to causephenotypic abnormalities in amphibian embryos such as those disclosedherein (e.g., split tail, abnormal head development, lack of mesodermdevelopment upon FGF-induction, failure of dorsal closure, and thelike), to exhibit increased expression only in injured vessel adventitiabut not in uninjured vessels nor in the neointima of injured oruninjured vessels, the ability to induce adventitial cell proliferation,to be inhibited by a soluble TGF-β receptor II (which blocks TGF-βsignaling), the ability to be induced in fibroblasts during woundhealing, the ability to be expressed by osteoblasts during boneformation, the ability to be expressed in osteoblasts adjacent tomineralized bone, the ability to be strongly expressed along fullthickness skin incisions undergoing wound healing and remodeling, theability to mediate cell death in endothelial cells when overexpressed,the ability to inhibit cell adhesion and cell-cell interaction when anantisense nucleic complementary to the nucleic acid encoding themolecule is expressed in a cell, and the ability to mediate excessive orinsufficient wound healing responses, scarring, keloids, bone formation,bone density, lack of dorsal closure, spina bifida-like effects,fracture healing, and the like.

[0257] A “restriction site” is a portion of a double-stranded nucleicacid which is recognized by a restriction endonuclease.

[0258] A portion of a double-stranded nucleic acid is “recognized” by arestriction endonuclease if the endonuclease is capable of cleaving bothstrands of the nucleic acid at the portion when the nucleic acid and theendonuclease are contacted.

[0259] By the term “specifically binds,” as used herein, is meant acompound, e.g., a protein, a nucleic acid, an antibody, and the like,which recognizes and binds a specific molecule, but does notsubstantially recognize or bind other molecules in a sample.

[0260] A first oligonucleotide anneals with a second oligonucleotide“with high stringency” if the two oligonucleotides anneal underconditions whereby only oligonucleotides which are at least about 73%,more preferably, at least about 75%, even more preferably, at leastabout 80%, even more preferably, at least about 85%, yet morepreferably, at least about 90%, and most preferably, at least about 95%,complementary anneal with one another. The stringency of conditions usedto anneal two oligonucleotides is a function of, among other factors,temperature, ionic strength of the annealing medium, the incubationperiod, the length of the oligonucleotides, the G-C content of theoligonucleotides, and the expected degree of non-homology between thetwo oligonucleotides, if known. Methods of adjusting the stringency ofannealing conditions are known (see, e.g., Sambrook et al., 1989,Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory,New York).

[0261] As used herein, the term “transgene” means an exogenous nucleicacid sequence which exogenous nucleic acid is encoded by a transgeniccell or mammal.

[0262] A “recombinant cell” is a cell that comprises a transgene. Such acell may be a eukaryotic cell or a prokaryotic cell. Also, thetransgenic cell encompasses, but is not limited to, an embryonic stemcell comprising the transgene, a cell obtained from a chimeric mammalderived from a transgenic ES cell where the cell comprises thetransgene, a cell obtained from a transgenic mammal, or fetal orplacental tissue thereof, and a prokaryotic cell comprising thetransgene.

[0263] By the term “exogenous nucleic acid” is meant that the nucleicacid has been introduced into a cell or an animal using technology whichhas been developed for the purpose of facilitating the introduction of anucleic acid into a cell or an animal.

[0264] By “tag” polypeptide is meant any protein which, when linked by apeptide bond to a protein of interest, may be used to localize theprotein, to purify it from a cell extract, to immobilize it for use inbinding assays, or to otherwise study its biological properties and/orfunction.

[0265] “TGF-β signaling”, as the term is used herein is any signalmediated by TGF-β interaction with a cognate receptor, e.g., TGF-βreceptor type II. One result of such interaction is an increase inREMODELIN receptor in a cell comprising the receptor on its surface.Other effects of TGF-β signaling are well-known in the art and can beassessed using standard methods also known in the relevant art.

[0266] As used herein, the term “transgenic mammal” means a mammal, thegerm cells of which comprise an exogenous nucleic acid.

[0267] As used herein, to “treat” means reducing the frequency withwhich symptoms of arterial restenosis, adventitial fibrosis, excessiveor insufficient wound healing responses, scarring, keloids, boneformation, fracture healing, and the like, are experienced by a patient.

[0268] By the term “vector” as used herein, is meant any plasmid orvirus encoding an exogenous nucleic acid. The term should also beconstrued to include non-plasmid and non-viral compounds whichfacilitate transfer of nucleic acid into virions or cells, such as, forexample, polylysine compounds and the like. The vector may be a viralvector which is suitable as a delivery vehicle for delivery of theREMODELIN protein or nucleic acid encoding a mammalian REMODELIN, to thepatient, or the vector may be a non-viral vector which is suitable forthe same purpose.

[0269] Examples of viral and non-viral vectors for delivery of DNA tocells and tissues are well known in the art and are described, forexample, in Ma et al. (1997, Proc. Natl. Acad. Sci. U.S.A.94:12744-12746). Examples of viral vectors include, but are not limitedto, a recombinant vaccinia virus, a recombinant adenovirus, arecombinant retrovirus, a recombinant adeno-associated virus, arecombinant avian pox virus, and the like (Cranage et al., 1986, EMBO J.5:3057-3063; International Patent Application No. WO94/17810, publishedAug. 18, 1994; International Patent Application No. WO94/23744,published Oct. 27, 1994). Examples of non-viral vectors include, but arenot limited to, liposomes, polyamine derivatives of DNA, and the like.

[0270] A “knock-out targeting vector,” as the term is used herein, meansa vector comprising two nucleic acid sequences each of which iscomplementary to a nucleic acid regions flanking a target sequence ofinterest which is to be deleted and/or replaced by another nucleic acidsequence. The two nucleic acid sequences therefore flank the targetsequence which is to be removed by the process of homologousrecombination

[0271] Description

[0272] I. Isolated Nucleic Acids

[0273] A. Sense Nucleic Acids

[0274] The present invention includes an isolated nucleic acid encodinga mammalian adventitia-inducible and bone expressed molecule, REMODELIN,or a fragment thereof, wherein the nucleic acid shares at least about33% identity with at least one nucleic acid having the sequence of(SEQID NO: 1) and (SEQ ID NO: 3). Preferably, the nucleic acid is at leastabout 35% homologous, more preferably, at least about 40% homologous,even more preferably, at least about 45% homologous, yet morepreferably, at least about 50% homologous, more preferably, at leastabout 55% homologous, more preferably, at least about 60% homologous,even more preferably, at least about 65% homologous, yet morepreferably, at least about 70% homologous, more preferably, at leastabout 75% homologous, even more preferably, at least about 80%homologous, yet more preferably, at least about 85% homologous, morepreferably, at least about 90% homologous, even more preferably, atleast about 95% homologous, and most preferably, at least about 99%homologous to at least one of SEQ ID NO: 1 and SEQ ID NO: 3 disclosedherein. Even more preferably, the nucleic acid is at least one of SEQ IDNO: 1 and SEQ ID NO: 3.

[0275] The present invention includes an isolated nucleic acid encodingrat REMODELIN, or a fragment thereof, wherein the nucleic acid shares atleast about 33% homology with a nucleic acid having the sequence SEQ IDNO: 1. Preferably, the nucleic acid is at least about 35% homologous,more preferably, at least about 40% homologous, even more preferably, atleast about 45% homologous, yet more preferably, at least about 50%homologous, more preferably, at least about 55% homologous, morepreferably, at least about 60% homologous, even more preferably, atleast about 65% homologous, yet more preferably, at least about 70%homologous, more preferably, at least about 75% homologous, even morepreferably, at least about 80% homologous, yet more preferably, at leastabout 85% homologous, more preferably, at least about 90% homologous,even more preferably, at least about 95% homologous, and mostpreferably, at least about 99% homologous to the rat REMODELIN disclosedherein (SEQ ID NO: 1). Even more preferably, the nucleic acid is SEQ IDNO: 1.

[0276] The present invention includes an isolated nucleic acid encodinghuman REMODELIN, or a fragment thereof, wherein the nucleic acid sharesat least about 33% homology with human REMODELIN (SEQ ID NO: 3).Preferably, the nucleic acid is at least about 35% homologous, morepreferably, at least about 40% homologous, even more preferably, atleast about 45% homologous, yet more preferably, at least about 50%homologous, more preferably, at least about 55% homologous, morepreferably, at least about 60% homologous, even more preferably, atleast about 65% homologous, yet more preferably, at least about 70%homologous, more preferably, at least about 75% homologous, even morepreferably, at least about 80% homologous, yet more preferably, at leastabout 85% homologous, more preferably, at least about 90% homologous,even more preferably, at least about 95% homologous, and mostpreferably, at least about 99% homologous to the human REMODELINdisclosed herein (SEQ ID NO: 3). Even more preferably, the nucleic acidis SEQ ID NO: 3.

[0277] In another aspect, the present invention includes an isolatednucleic acid encoding a mammalian REMODELIN, or a fragment thereof,wherein the protein encoded by the nucleic acid shares greater thanabout 6% homology with the amino acid sequence of at least one of SEQ IDNO: 2 (rat REMODELIN_(S)), SEQ ID NO: 4 (human REMODELIN), and SEQ IDNO: 5 (rat REMODELIN_(L)). That is, searching GenBank databasesdisclosed that REMODELIN shares about 62% sequence identity with aportion of the sequence GenBank Acc. No. P27393, collagen alpha-2 (IV)chain precursor, over a stretch of about 35 amino acids. Full-lengthREMODELIN protein comprises about 243 amino acids such that full-lengthREMODELIN shares about 5.7% overall sequence identity with collagenalpha-2 (IV) chain precursor (i.e., GenBank Acc. No. P27393).

[0278] Preferably, the protein encoded by the isolated nucleic acidencoding REMODELIN is at least about 10% homologous, more preferably, atleast about 15% homologous, more preferably, at least about 20%homologous, even more preferably, at least about 25% homologous, morepreferably, at least about 30% homologous, preferably, at least about35% homologous, even more preferably, at least about 40% homologous,even more preferably, at least about 45% homologous, yet morepreferably, at least about 50% homologous, more preferably, at leastabout 55% homologous, more preferably, at least about 60% homologous,even more preferably, at least about 65% homologous, yet morepreferably, at least about 70% homologous, more preferably, at leastabout 75% homologous, even more preferably, at least about 80%homologous, yet more preferably, at least about 85% homologous, morepreferably, at least about 90% homologous, even more preferably, atleast about 95% homologous, and most preferably, at least about 99%homologous to at least one of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:5. Even more preferably, the REMODELIN protein encoded by the nucleicacid is at least one of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.

[0279] In another aspect, the present invention includes an isolatednucleic acid encoding rat REMODELIN, or a fragment thereof, wherein theprotein encoded by the nucleic acid shares at least about 6% homologywith the amino acid sequence of SEQ ID NO: 2. Preferably, the proteinencoded by the isolated nucleic acid encoding REMODELIN is at leastabout 10% homologous, more preferably, at least about 15% homologous,more preferably, at least about 20% homologous, even more preferably, atleast about 25% homologous, more preferably, at least about 30%homologous, preferably, at least about 35% homologous, even morepreferably, at least about 40% homologous, even more preferably, atleast about 45% homologous, yet more preferably, at least about 50%homologous, more preferably, at least about 55% homologous, morepreferably, at least about 60% homologous, even more preferably, atleast about 65% homologous, yet more preferably, at least about 70%homologous, more preferably, at least about 75% homologous, even morepreferably, at least about 80% homologous, yet more preferably, at leastabout 85% homologous, more preferably, at least about 90% homologous,even more preferably, at least about 95% homologous, and mostpreferably, at least about 99% homologous to rat REMODELIN disclosedherein (SEQ ID NO: 2). Even more preferably, the rat REMODELIN proteinencoded by the nucleic acid is SEQ ID NO: 2.

[0280] In another aspect, the present invention includes an isolatednucleic acid encoding human REMODELIN, or a fragment thereof, whereinthe protein encoded by the nucleic acid shares at least about 6%homology with the amino acid sequence of SEQ ID NO: 4. Preferably, theprotein encoded by the isolated nucleic acid encoding REMODELIN is atleast about 10% homologous, more preferably, at least about 15%homologous, more preferably, at least about 20% homologous, even morepreferably, at least about 25% homologous, more preferably, at leastabout 30% homologous, preferably, at least about 35% homologous, evenmore preferably, at least about 40% homologous, even more preferably, atleast about 45% homologous, yet more preferably, at least about 50%homologous, more preferably, at least about 55% homologous, morepreferably, at least about 60% homologous, even more preferably, atleast about 65% homologous, yet more preferably, at least about 70%homologous, more preferably, at least about 75% homologous, even morepreferably, at least about 80% homologous, yet more preferably, at leastabout 85% homologous, more preferably, at least about 90% homologous,even more preferably, at least about 95% homologous, and mostpreferably, at least about 99% homologous to the human REMODELINdisclosed herein (SEQ ID NO: 4). Even more preferably, the humanREMODELIN protein encoded by the nucleic acid is SEQ ID NO: 4.

[0281] One skilled in the art would understand, based upon thedisclosure provided herein, that a nucleic acid encoding a rat REMODELIN(SEQ ID NO: 1) can be alternatively translated to produce an alternaterat REMODELIN protein comprising 245 amino acids (rat REMODELIN_(S); SEQID NO: 2) and a protein comprising an additional 32 amino acid residuesat the N-terminus (i.e., the 277 amino acid long form of REMODELINdesignated REMODELIN_(L) [SEQ ID NO: 5]) since the nucleic acid encodingrat REMODELIN (SEQ ID NO: 1) comprises two putative transcriptionalstart sites at positions 19 and 1 16 (FIG. 4A) that are compatible withthe Kozak rule.

[0282] Therefore, in another aspect, the present invention includes anisolated nucleic acid encoding rat REMODELIN, or a fragment thereof,wherein the protein encoded by the nucleic acid shares at least about 6%homology with the amino acid sequence of SEQ ID NO: 5 (i.e., 277 aminoacid rat REMODELIN_(L)). Preferably, the protein encoded by the isolatednucleic acid encoding REMODELIN is at least about 10% homologous, morepreferably, at least about 15% homologous, more preferably, at leastabout 20% homologous, even more preferably, at least about 25%homologous, more preferably, at least about 30% homologous, preferably,at least about 35% homologous, even more preferably, at least about 40%homologous, even more preferably, at least about 45% homologous, yetmore preferably, at least about 50% homologous, more preferably, atleast about 55% homologous, more preferably, at least about 60%homologous, even more preferably, at least about 65% homologous, yetmore preferably, at least about 70% homologous, more preferably, atleast about 75% homologous, even more preferably, at least about 80%homologous, yet more preferably, at least about 85% homologous, morepreferably, at least about 90% homologous, even more preferably, atleast about 95% homologous, and most preferably, at least about 99%homologous to the rat REMODELIN_(L) disclosed herein (SEQ ID NO: 5).Even more preferably, the rat REMODELIN_(L) protein encoded by thenucleic acid is SEQ ID NO: 5.

[0283] One skilled in the art would appreciate, based upon thedisclosure provided herein, that a mouse REMODELIN homolog likely existsand can be readily identified and isolated using the methods describedherein using the sequence data disclosed herein regarding thehighly-conserved rat and mouse homologs. Thus, the present inventionencompasses additional REMODELINs that can be readily identified basedupon the disclosure provided herein, including, but not limited to,mouse REMODELIN.

[0284] The isolated nucleic acid of the invention should be construed toinclude an RNA or a DNA sequence encoding a REMODELIN protein of theinvention (excluding the isolated nucleic acids referred to by thefollowing GenBank Accession Nos: AA335862; C01758; AA335551; AA406425;R46857; AA584310; D79314; AI085616; D62262; AA482398; AA482544;AI359844; AI352209; AI239604; AI218433; AI081084; AI074870; AI074769;AA974239; AA969841; AA857920; AA723450; AA410434; AA738416; AI370649;AA507081), and any modified forms thereof, including chemicalmodifications of the DNA or RNA which render the nucleotide sequencemore stable when it is cell free or when it is associated with a cell.Chemical modifications of nucleotides may also be used to enhance theefficiency with which a nucleotide sequence is taken up by a cell or theefficiency with which it is expressed in a cell. Any and allcombinations of modifications of the nucleotide sequences arecontemplated in the present invention.

[0285] The present invention should not be construed as being limitedsolely to the nucleic and amino acid sequences disclosed herein. Oncearmed with the present invention, it is readily apparent to one skilledin the art that other nucleic acids encoding REMODELIN proteins can suchas those present in other species of mammals (e.g., ape, gibbon, bovine,ovine, equine, porcine, canine, feline, and the like) be obtained byfollowing the procedures described herein in the experimental detailssection for the isolation of the rat, and human REMODELIN nucleic acidsencoding REMODELIN polypeptides as disclosed herein (e.g., screening ofgenomic or cDNA libraries), and procedures that are well-known in theart (e.g., reverse transcription PCR using mRNA samples) or to bedeveloped.

[0286] Further, any number of procedures may be used for the generationof mutant, derivative or variant forms of REMODELIN using recombinantDNA methodology well known in the art such as, for example, thatdescribed in Sambrook et al. (1989, Molecular Cloning: A LaboratoryManual, Cold Spring Harbor Laboratory Press, New York) and Ausubel etal. (1997, Current Protocols in Molecular Biology, Green & Wiley, NewYork).

[0287] Procedures for the introduction of amino acid changes in aprotein or polypeptide by altering the DNA sequence encoding thepolypeptide are well known in the art and are also described in Sambrooket al. (1989, supra); Ausubel et al. (1997, supra).

[0288] The invention includes a nucleic acid encoding a mammalianREMODELIN wherein a nucleic acid encoding a tag polypeptide iscovalently linked thereto. That is, the invention encompasses a chimericnucleic acid wherein the nucleic acid sequences encoding a tagpolypeptide is covalently linked to the nucleic acid encoding at leastone of rat REMODELIN and human REMODELIN. Such tag polypeptides are wellknown in the art and include, for instance, green fluorescent protein(GFP), an influenza virus hemagglutinin tag polypeptide, myc,myc-pyruvate kinase (myc-PK), His₆, maltose biding protein (MBP), a FLAGtag polypeptide, and a glutathione-S-transferase (GST) tag polypeptide.However, the invention should in no way be construed to be limited tothe nucleic acids encoding the above-listed tag polypeptides. Rather,any nucleic acid sequence encoding a polypeptide which may function in amanner substantially similar to these tag polypeptides should beconstrued to be included in the present invention.

[0289] The nucleic acid comprising a nucleic acid encoding a tagpolypeptide can be used to localize REMODELIN within a cell, a tissue(e.g., a blood vessel, bone, and the like), and/or a whole organism(e.g., an amphibian and/or a mammalian embryo, and the like), detectREMODELIN if secreted from a cell, and to study the role(s) of REMODELINin a cell. Further, addition of a tag polypeptide facilitates isolationand purification of the “tagged” protein such that the proteins of theinvention can be produced and purified readily.

[0290] B. Antisense Nucleic Acids

[0291] In certain situations, it may be desirable to inhibit expressionof REMODELIN and the invention therefore includes compositions usefulfor inhibition of REMODELIN expression. Thus, the invention features anisolated nucleic acid complementary to a portion or all of a nucleicacid encoding a mammalian REMODELIN, which nucleic acid is in anantisense orientation with respect to transcription. Preferably, theantisense nucleic acid is complementary with a nucleic acid having atleast about 33% homology with at least one of SEQ ID NO: 1 and SEQ IDNO: 3, or a fragment thereof. Preferably, the nucleic acid is at leastabout 35% homologous, more preferably, at least about 40% homologous,even more preferably, at least about 45% homologous, yet morepreferably, at least about 50% homologous, more preferably, at leastabout 55% homologous, more preferably, at least about 60% homologous,even more preferably, at least about 65% homologous, yet morepreferably, at least about 70% homologous, more preferably, at leastabout 75% homologous, even more preferably, at least about 80%homologous, yet more preferably, at least about 85% homologous, morepreferably, at least about 90% homologous, even more preferably, atleast about 95% homologous, and most preferably, at least about 99%homologous to a nucleic acid complementary to a portion or all of anucleic acid encoding a mammalian REMODELIN having the sequence of atleast one of SEQ ID NO: 1 and SEQ ID NO: 3, or a fragment thereof, whichis in an antisense orientation with respect to transcription. Mostpreferably, the nucleic acid is complementary to a portion or all of anucleic acid that is at least one of SEQ ID NO: 1 and SEQ ID NO: 3, or afragment thereof. Such antisense nucleic acid serves to inhibit theexpression, function, or both, of an adventitia-inducible and boneexpressed (REMODELIN) molecule.

[0292] In one aspect, the invention includes an isolated nucleic acidcomplementary to a portion or all of a nucleic acid encoding a mammalianREMODELIN molecule, which nucleic acid is in an antisense orientationwith respect to transcription. Preferably, the antisense nucleic acid iscomplementary with a nucleic acid having at least about 33% homologywith SEQ ID NO: 1, or a fragment thereof. Preferably, the antisensenucleic acid is complementary with a nucleic acid having at least about35% identity, more preferably, at least about 40% identity, even morepreferably, at least about 45% identity, yet more preferably, at leastabout 50% identity, more preferably, at least about 55% identity, morepreferably, at least about 60% identity, even more preferably, at leastabout 65% identity, yet more preferably, at least about 70% identity,more preferably, at least about 75% identity, even more preferably, atleast about 80% identity, yet more preferably, at least about 85%identity, more preferably, at least about 90% identity, even morepreferably, at least about 95% identity, and most preferably, at leastabout 99% identity with a nucleic acid complementary to a portion or allof a nucleic acid encoding a mammalian REMODELIN having the sequence SEQID NO: 1

[0293] Most preferably, the nucleic acid is complementary to a portionor all of a nucleic acid that is SEQ ID NO: 1, or a fragment thereof.Such antisense nucleic acid serves to inhibit the expression, function,or both, of an adventitia-inducible and bone expressed REMODELINmolecule.

[0294] In another aspect, the invention includes an isolated nucleicacid complementary to a portion or all of a nucleic acid encoding amammalian REMODELIN molecule, which nucleic acid is in an antisenseorientation with respect to transcription. Preferably, the antisensenucleic acid is complementary with a nucleic acid having at least about35% identity, more preferably, at least about 40% identity, even morepreferably, at least about 45% identity, yet more preferably, at leastabout 50% identity, more preferably, at least about 55% identity, morepreferably, at least about 60% identity, even more preferably, at leastabout 65% identity, yet more preferably, at least about 70% identity,more preferably, at least about 75% identity, even more preferably, atleast about 80% identity, yet more preferably, at least about 85%identity, more preferably, at least about 90% identity, even morepreferably, at least about 95% identity, and most preferably, at leastabout 99% identity with a nucleic acid complementary to a nucleic acidencoding a mammalian REMODELIN having the sequence SEQ ID NO: 3, or afragment thereof. Most preferably, the nucleic acid is complementary toa portion or all of a nucleic acid that is SEQ ID NO: 3, or a fragmentthereof. Such antisense nucleic acid serves to inhibit the expression,function, or both, of an adventitia-inducible and bone expressedREMODELIN molecule.

[0295] Further, antisense nucleic acids complementary to all or aportion of a nucleic acid encoding REMODELIN can be used to detect theexpression of REMODELIN mRNA in a cell, tissue, and/or organism, using,for example but not limited to, in situ hybridization. Thus, one skilledin the art would understand, based upon the disclosure provided herein,that the invention encompasses antisense nucleic acids that can be usedas probes to assess REMODELIN expression. Such antisense nucleic acidsencompass, but are not limited to, a nucleic acid having the sequenceSEQ ID NO: 6.

[0296] Antisense molecules of the invention may be made syntheticallyand then provided to the cell. Antisense oligomers of between about 10to about 30, and more preferably about 15 nucleotides, are preferred,since they are easily synthesized and introduced into a target cell.Synthetic antisense molecules contemplated by the invention includeoligonucleotide derivatives known in the art which have improvedbiological activity compared to unmodified oligonucleotides (see Cohen,supra; Tullis, 1991, U.S. Pat. No. 5,023,243, incorporated by referenceherein in its entirety).

[0297] II. Isolated Polypeptides

[0298] The invention also includes an isolated polypeptide comprising amammalian REMODELIN. Preferably, the isolated polypeptide comprising amammalian REMODELIN is at least about 6% homologous to a polypeptidehaving the amino acid sequence of at least one of SEQ ID NO: 2, SEQ IDNO: 4, and SEQ ID NO: 5. Preferably, the isolated polypeptide is atleast about 10% homologous, more preferably, at least about 15%homologous, more preferably, at least about 20% homologous, even morepreferably, at least about 25% homologous, more preferably, at leastabout 30% homologous, preferably, at least about 35% homologous, evenmore preferably, at least about 40% homologous, even more preferably, atleast about 45% homologous, yet more preferably, at least about 50%homologous, more preferably, at least about 55% homologous, morepreferably, at least about 60% homologous, even more preferably, atleast about 65% homologous, yet more preferably, at least about 70%homologous, more preferably, at least about 75% homologous, even morepreferably, at least about 80% homologous, yet more preferably, at leastabout 85% homologous, more preferably, at least about 90% homologous,even more preferably, at least about 95% homologous, and mostpreferably, at least about 99% homologous to at least one of SEQ ID NO:2, SEQ ID NO: 4, and SEQ ID NO: 5. More preferably, the isolatedpolypeptide comprising a mammalian REMODELIN is at least one of ratREMODELIN_(S), human REMODELIN, and rat REMODELIN_(L). Most preferably,the isolated polypeptide comprising a mammalian REMODELIN is at leastone of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.

[0299] The invention also includes an isolated polypeptide comprising amammalian REMODELIN molecule. Preferably, the isolated polypeptidecomprising a mammalian REMODELIN is at least about 6% homologous to apolypeptide having the amino acid sequence of SEQ ID NO: 2. Morepreferably, the isolated polypeptide comprising a mammalian REMODELIN isat least about 10% homologous, more preferably, at least about 15%homologous, more preferably, at least about 20% homologous, even morepreferably, at least about 25% homologous, more preferably, at leastabout 30% homologous, preferably, at least about 35% homologous, evenmore preferably, at least about 40% homologous, even more preferably, atleast about 45% homologous, yet more preferably, at least about 50%homologous, more preferably, at least about 55% homologous, morepreferably, at least about 60% homologous, even more preferably, atleast about 65% homologous, yet more preferably, at least about 70%homologous, more preferably, at least about 75% homologous, even morepreferably, at least about 80% homologous, yet more preferably, at leastabout 85% homologous, more preferably, at least about 90% homologous,even more preferably, at least about 95% homologous, and mostpreferably, at least about 99% homologous to rat REMODELIN_(S) (SEQ IDNO: 2) More preferably, the isolated polypeptide comprising a mammalianREMODELIN is rat REMODELIN_(S). Most preferably, the isolatedpolypeptide comprising a mammalian REMODELIN molecule is SEQ ID NO: 2.

[0300] The invention also includes an isolated polypeptide comprising amammalian REMODELIN molecule. Preferably, the isolated polypeptidecomprising a mammalian REMODELIN molecule is at least about 6%homologous to a polypeptide having the amino acid sequence of SEQ ID NO:4. More preferably, the isolated polypeptide comprising a mammalianREMODELIN is at least is at least about 10% homologous, more preferably,at least about 15% homologous, more preferably, at least about 20%homologous, even more preferably, at least about 25% homologous, morepreferably, at least about 30% homologous, preferably, at least about35% homologous, even more preferably, at least about 40% homologous,even more preferably, at least about 45% homologous, yet morepreferably, at least about 50% homologous, more preferably, at leastabout 55% homologous, more preferably, at least about 60% homologous,even more preferably, at least about 65% homologous, yet morepreferably, at least about 70% homologous, more preferably, at leastabout 75% homologous, even more preferably, at least about 80%homologous, yet more preferably, at least about 85% homologous, morepreferably, at least about 90% homologous, even more preferably, atleast about 95% homologous, and most preferably, at least about 99%homologous to SEQ ID NO: 4. More preferably, the isolated polypeptidecomprising a mammalian REMODELIN molecule is human REMODELIN. Mostpreferably, the isolated polypeptide comprising a mammalian REMODELINmolecule is SEQ ID NO: 4.

[0301] The invention also includes an isolated polypeptide comprising amammalian REMODELIN molecule. Preferably, the isolated polypeptidecomprising a mammalian REMODELIN molecule is at least about 6%homologous to a polypeptide having the amino acid sequence of SEQ ID NO:5. More preferably, the isolated polypeptide comprising a mammalianREMODELIN is at least about 10% homologous, more preferably, at leastabout 15% homologous, more preferably, at least about 20% homologous,even more preferably, at least about 25% homologous, more preferably, atleast about 30% homologous, preferably, at least about 35% homologous,even more preferably, at least about 40% homologous, even morepreferably, at least about 45% homologous, yet more preferably, at leastabout 50% homologous, more preferably, at least about 55% homologous,more preferably, at least about 60% homologous, even more preferably, atleast about 65% homologous, yet more preferably, at least about 70%homologous, more preferably, at least about 75% homologous, even morepreferably, at least about 80% homologous, yet more preferably, at leastabout 85% homologous, more preferably, at least about 90% homologous,even more preferably, at least about 95% homologous, and mostpreferably, at least about 99% homologous to rat REMODELIN_(L). Morepreferably, the isolated polypeptide comprising a mammalian REMODELINmolecule is rat REMODELIN_(L). Most preferably, the isolated polypeptidecomprising a mammalian REMODELIN molecule is SEQ ID NO: 5.

[0302] The present invention also provides for analogs of proteins orpeptides which comprise a REMODELIN as disclosed herein. Analogs maydiffer from naturally occurring proteins or peptides by conservativeamino acid sequence differences or by modifications which do not affectsequence, or by both. For example, conservative amino acid changes maybe made, which although they alter the primary sequence of the proteinor peptide, do not normally alter its function. Conservative amino acidsubstitutions typically include substitutions within the followinggroups:

[0303] glycine, alanine;

[0304] valine, isoleucine, leucine;

[0305] aspartic acid, glutamic acid;

[0306] asparagine, glutamine;

[0307] serine, threonine;

[0308] lysine, arginine;

[0309] phenylalanine, tyrosine.

[0310] Modifications (which do not normally alter primary sequence)include in vivo, or in vitro, chemical derivatization of polypeptides,e.g., acetylation, or carboxylation. Also included are modifications ofglycosylation, e.g., those made by modifying the glycosylation patternsof a polypeptide during its synthesis and processing or in furtherprocessing steps; e.g., by exposing the polypeptide to enzymes whichaffect glycosylation, e.g., mammalian glycosylating or deglycosylatingenzymes. Also embraced are sequences which have phosphorylated aminoacid residues, e.g., phosphotyrosine, phosphoserine, orphosphothreonine.

[0311] Also included are polypeptides which have been modified usingordinary molecular biological techniques so as to improve theirresistance to proteolytic degradation or to optimize solubilityproperties or to render them more suitable as a therapeutic agent.Analogs of such polypeptides include those containing residues otherthan naturally occurring L-amino acids, e.g., D-amino acids ornon-naturally occurring synthetic amino acids. The peptides of theinvention are not limited to products of any of the specific exemplaryprocesses listed herein.

[0312] The present invention should also be construed to encompass“mutants,” “derivatives,” and “variants” of the peptides of theinvention (or of the DNA encoding the same) which mutants, derivativesand variants are REMODELIN peptides which are altered in one or moreamino acids (or, when referring to the nucleotide sequence encoding thesame, are altered in one or more base pairs) such that the resultingpeptide (or DNA) is not identical to the sequences recited herein, buthas the same biological property as the peptides disclosed herein, inthat the peptide has biological/biochemical properties of the REMODELINpeptide of the present invention.

[0313] A “biological property of a REMODELIN protein’ should beconstrued but not be limited to include, the ability of the expressionof the peptide to be induced by TGF-β, the ability of the peptide to beexpressed selectively in adventitia, the ability of the peptide to beinduced by balloon-injury, the ability of the peptide to be expressed inbone, the ability of the peptide to be expressed in a mouse embryocommencing at about day 11.5 post coitus, the ability of a molecule tobe selectively induced in adventitia of injured vessels, to causephenotypic abnormalities in amphibian embryos such as those disclosedherein (e.g., split tail, abnormal head development, lack of mesodermdevelopment upon FGF-induction, failure of dorsal closure, and thelike), to exhibit increased expression only in injured vessel adventitiabut not in uninjured vessels nor in the neointima of injured oruninjured vessels, the ability to induce adventitial cell proliferation,to be inhibited by a soluble TGF-β receptor II (which blocks TGF-βsignaling), the ability to be induced in fibroblasts during woundhealing, the ability to be expressed by osteoblasts during boneformation, the ability to mediate cell death in endothelial cells whenoverexpressed, the ability to affect cell adhesion and cell-cellinteraction, the ability to affect bone density and/or bone growth, andthe ability to mediate excessive or insufficient wound healingresponses, scarring, keloids, bone formation, fracture healing, and thelike.

[0314] Further, the invention should be construed to include naturallyoccurring variants or recombinantly derived mutants of REMODELINsequences, which variants or mutants render the protein encoded therebyeither more, less, or just as biologically active as the full-lengthclones of the invention.

[0315] The nucleic acids, and peptides encoded thereby, are useful toolsfor elucidating the function(s) of REMODELIN molecule in a cell.Further, nucleic and amino acids comprising mammalian REMODELIN moleculeare useful diagnostics which can be used, for example, to identify acompound that affects REMODELIN expression and/or TGF-β signaling, andthe like, and is a potential therapeutic drug candidate for arterialrestenosis, anti-cancer therapy, to promote or inhibit wound healing, toinhibit scar tissue or keloid formation, to promote bone fracturehealing, and the like. The nucleic acids, the proteins encoded thereby,or both, can be administered to a mammal to increase or decreaseexpression of REMODELIN in the mammal. This can be beneficial for themammal in situations where under or over-expression of REMODELIN in themammal mediates a disease or condition associated with alteredexpression of REMODELIN compared with normal expression of REMODELIN ina healthy mammal. Such conditions that can be affected by modulatingREMODELIN expression thereby providing a therapeutic benefit include,but are not limited to, wound healing, arterial injury, ossification,and the like. This is because, as more fully disclosed elsewhere herein,REMODELIN is transiently expressed in (myo)fibroblasts in conditionsassociated with healing and repair following tissue injury. Forinstance, REMODELIN is expressed in osteoblasts bone, which isundergoing constant remodeling. Additionally, over-expression ofREMODELIN during embryogenesis affects dorsal closure, bone density, andbone growth, and mediates and/or is associated with spina bifida-likeeffects all of which demonstrate the important biological role(s) ofREMODELIN.

[0316] Additionally, the nucleic and amino acids of the invention can beused to produce recombinant cells and transgenic non-human mammals whichare useful tools for the study of REMODELIN action, the identificationof novel diagnostics and therapeutics for treatment, and for elucidatingthe cellular role(s) of REMODELIN, among other things. For instance,transgenic animals can be used to study bone related, wound healingrelated, and vascular disease related conditions.

[0317] Further, the nucleic and amino acids of the invention can be useddiagnostically, either by assessing the level of gene expression orprotein expression, to assess severity and prognosis of negativeremodeling, arterial restenosis, vessel injury, fibrosis, bone growth,and the like. The nucleic acids and proteins of the invention are alsouseful in the development of assays to assess the efficacy of atreatment for preventing arterial restenosis, affecting bone density andbone growth, and the like. That is, the nucleic acids and polypeptidesof the invention can be used to detect the effect of various therapieson REMODELIN expression, thereby ascertaining the effectiveness of thetherapies such as, but not limited to, assessment of treatmentefficacies for restenosis, anti-fibrotic therapy in any tissue,therapies to promote wound healing in any tissue and therapies for boneformation including bone fracture healing.

[0318] III. Vectors

[0319] In other related aspects, the invention includes an isolatednucleic acid encoding a mammalian REMODELIN operably linked to a nucleicacid comprising a promoter/regulatory sequence such that the nucleicacid is preferably capable of directing expression of the proteinencoded by the nucleic acid. Thus, the invention encompasses expressionvectors and methods for the introduction of exogenous DNA into cellswith concomitant expression of the exogenous DNA in the cells such asthose described, for example, in Sambrook et al. (1989, supra), andAusubel et al. (1997, supra).

[0320] Expression of REMODELIN, either alone or fused to a detectabletag polypeptide, in cells which either do not normally express theREMODELIN or which do not express REMODELIN fused with a tagpolypeptide, may be accomplished by generating a plasmid, viral, orother type of vector comprising the desired nucleic acid operably linkedto a promoter/regulatory sequence which serves to drive expression ofthe protein, with or without tag, in cells in which the vector isintroduced. Many promoter/regulatory sequences useful for drivingconstitutive expression of a gene are available in the art and include,but are not limited to, for example, the cytomegalovirus immediate earlypromoter enhancer sequence, the SV40 early promoter, both of which wereused in the experiments disclosed herein, as well as the Rous sarcomavirus promoter, and the like. Moreover, inducible and tissue specificexpression of the nucleic acid encoding REMODELIN may be accomplished byplacing the nucleic acid encoding REMODELIN, with or without a tag,under the control of an inducible or tissue specific promoter/regulatorysequence. Examples of tissue specific or inducible promoter/regulatorysequences which are useful for his purpose include, but are not limitedto the MMTV LTR inducible promoter, and the SV40 late enhancer/promoter.In addition, promoters which are well known in the art which are inducedin response to inducing agents such as metals, glucocorticoids, and thelike, are also contemplated in the invention. Thus, it will beappreciated that the invention includes the use of anypromoter/regulatory sequence, which is either known or unknown, andwhich is capable of driving expression of the desired protein operablylinked thereto.

[0321] Expressing REMODELIN using a vector allows the isolation of largeamounts of recombinantly produced protein. Further, where the lack ordecreased level of REMODELIN expression causes a disease, disorder, orcondition associated with such expression, the expression of REMODELINdriven by a promoter/regulatory sequence can provide useful therapeuticsincluding, but not limited to, gene therapy whereby REMODELIN isprovided. A disease, disorder or condition associated with a decreasedlevel of expression, level of protein, or decreased activity of theprotein, for which administration of REMODELIN can be useful caninclude, but is not limited to, bone formation, bone fracture healing,wound healing and repair in any tissue, and the like. Therefore, theinvention includes not only methods of inhibiting REMODELIN expression,translation, and/or activity, but it also includes methods relating toincreasing REMODELIN expression, protein level, and/or activity sinceboth decreasing and increasing REMODELIN expression and/or activity canbe useful in providing effective therapeutics.

[0322] One skilled in the art would appreciate, based upon thedisclosure provided herein, that because of the selective expression ofREMODELIN during wound healing in response to injury in any tissue, thepromoter for REMODELIN can be an excellent choice for targeting nucleicacid expression of a desired gene to a site of tissue injury.

[0323] Selection of any particular plasmid vector or other DNA vector isnot a limiting factor in this invention and a wide plethora vectors iswell-known in the art. Further, it is well within the skill of theartisan to choose particular promoter/regulatory sequences and operablylink those promoter/regulatory sequences to a DNA sequence encoding adesired polypeptide. Such technology is well known in the art and isdescribed, for example, in Sambrook, supra, and Ausubel, supra.

[0324] The invention thus includes a vector comprising an isolatednucleic acid encoding a mammalian REMODELIN. The incorporation of adesired nucleic acid into a vector and the choice of vectors iswell-known in the art as described in, for example, Sambrook et al.,supra, and Ausubel et al., supra.

[0325] The invention also includes cells, viruses, proviruses, and thelike, containing such vectors. Methods for producing cells comprisingvectors and/or exogenous nucleic acids are well-known in the art. See,e.g., Sambrook et al., supra; Ausubel et al., supra.

[0326] The nucleic acids encoding REMODELIN can be cloned into variousplasmid vectors. However, the present invention should not be construedto be limited to plasmids or to any particular vector. Instead, thepresent invention should be construed to encompass a wide plethora ofvectors which are readily available and/or well-known in the art and novector at all.

[0327] IV. Antisense Molecules and Ribozymes

[0328] Further, the invention includes a recombinant cell comprising anantisense nucleic acid which cell is a useful model for elucidating therole(s) of REMODELIN in cellular processes. That is, the increasedexpression of REMODELIN in balloon-injured vessels and, morespecifically, in the adventitia thereof, indicate that REMODELIN isinvolved in cell proliferation associated with negative remodeling andarterial restenosis. Accordingly, a transgenic cell comprising anantisense nucleic acid complementary to REMODELIN but in an antisenseorientation is a useful tool for the study of the mechanism(s) of actionof REMODELIN and its role(s) in the cell and for the identification oftherapeutics that ameliorate the effect(s) of REMODELIN expression.

[0329] One skilled in the art can appreciate, based upon the disclosureprovided herein, that an antisense nucleic acid complementary to anucleic acid encoding REMODELIN can be used to transfect a cell and thecell can be studied to determine the effect(s) of altered expression ofREMODELIN in order to study the function(s) of REMODELIN and to identityuseful therapeutics and diagnostics.

[0330] Further, methods of decreasing REMODELIN expression and/oractivity in a cell can provide useful diagnostics and/or therapeuticsfor diseases, disorders or conditions mediated by or associated withincreased REMODELIN expression, increased level of REMODELIN protein ina cell or secretion therefrom, and/or increased REMODELIN activity. Suchdiseases, disorders or conditions include, but are not limited to, anycondition associated with fibrosis, e.g., proliferation of fibroblastswith or without excessive fibrous tissue formation, and any conditionassociated with excessive bone formation or ectopic ossification(malignant or benign), and the like.

[0331] One skilled in the art will appreciate that one way to decreasethe levels of REMODELIN mRNA and/or protein in a cell is to inhibitexpression of the nucleic acid encoding the protein. Expression ofREMODELIN may be inhibited using, for example, antisense molecules, andalso by using ribozymes or double-stranded RNA as described in, forexample, Wianny and Kernicka-Goetz (2000, Nature Cell Biol. 2:70-75).

[0332] Antisense molecules and their use for inhibiting gene expressionare well known in the art (see, e.g., Cohen, 1989, In:Oligodeoxyribonucleotides, Antisense Inhibitors of Gene Expression, CRCPress). Antisense nucleic acids are DNA or RNA molecules that arecomplementary, as that term is defined elsewhere herein, to at least aportion of a specific mRNA molecule (Weintraub, 1990, ScientificAmerican 262:40). In the cell, antisense nucleic acids hybridize to thecorresponding mRNA, forming a double-stranded molecule therebyinhibiting the translation of genes.

[0333] The use of antisense methods to inhibit the translation of genesis known in the art, and is described, for example, in Marcus-Sakura(1988, Anal. Biochem. 172:289). Such antisense molecules may be providedto the cell via genetic expression using DNA encoding the antisensemolecule as taught by Inoue (1993, U.S. Pat. No. 5,190,931).

[0334] Alternatively, antisense molecules of the invention may be madesynthetically and then provided to the cell. Antisense oligomers ofbetween about 10 to about 30, and more preferably about 15 nucleotides,are preferred, since they are easily synthesized and introduced into atarget cell. Synthetic antisense molecules contemplated by the inventioninclude oligonucleotide derivatives known in the art which have improvedbiological activity compared to unmodified oligonucleotides (see Cohen,supra; Tullis, 1991, U.S. Pat. No. 5,023,243, incorporated by referenceherein in its entirety).

[0335] Ribozymes and their use for inhibiting gene expression are alsowell known in the art (see, e.g., Cech et al., 1992, J. Biol. Chem.267:17479-17482; Hampel et al., 1989, Biochemistry 28:4929-4933;Eckstein et al., International Publication No. WO 92/07065; Altman etal., U.S. Pat. No. 5,168,053, incorporated by reference herein in itsentirety). Ribozymes are RNA molecules possessing the ability tospecifically cleave other single-stranded RNA in a manner analogous toDNA restriction endonucleases. Through the modification of nucleotidesequences encoding these RNAs, molecules can be engineered to recognizespecific nucleotide sequences in an RNA molecule and cleave it (Cech,1988, J. Amer. Med. Assn. 260:3030). A major advantage of this approachis that, because they are sequence-specific, only mRNAs with particularsequences are inactivated.

[0336] There are two basic types of ribozymes, namely, tetrahymena-type(Hasselhoff, 1988, Nature 334:585) and hammerhead-type. Tetrahymena-typeribozymes recognize sequences which are four bases in length, whilehammerhead-type ribozymes recognize base sequences 11-18 bases inlength. The longer the sequence, the greater the likelihood that thesequence will occur exclusively in the target mRNA species.Consequently, hammerhead-type ribozymes are preferable totetrahymena-type ribozymes for inactivating specific mRNA species, and18-base recognition sequences are preferable to shorter recognitionsequences which may occur randomly within various unrelated mRNAmolecules.

[0337] Ribozymes useful for inhibiting the expression of REMODELIN canbe designed by incorporating target sequences into the basic ribozymestructure which are complementary to the mRNA sequence of the REMODELINencoded by REMODELIN or having at least about 33% homology to at leastone of SEQ ID NO: 1 and SEQ ID NO: 3. Preferably, the sequence is atleast about 35% homologous, even more preferably, at least about 40%homologous, even more preferably, at least about 45% homologous, yetmore preferably, at least about 50% homologous, more preferably, atleast about 55% homologous, more preferably, at least about 60%homologous, even more preferably, at least about 65% homologous, yetmore preferably, at least about 70% homologous, more preferably, atleast about 75% homologous, even more preferably, at least about 80%homologous, yet more preferably, at least about 85% homologous, morepreferably, at least about 90% homologous, even more preferably, atleast about 95% homologous, and most preferably, at least about 99%homologous to at least one of SEQ ID NO: 1 and SEQ ID NO: 3. Ribozymestargeting REMODELIN may be synthesized using commercially availablereagents (Applied Biosystems, Inc., Foster City, Calif.) or they may begenetically expressed from DNA encoding them.

[0338] V. Recombinant Cells and Transgenic Non-human Mammals

[0339] The invention includes a recombinant cell comprising, inter alia,an isolated nucleic acid encoding REMODELIN, an antisense nucleic acidcomplementary thereto, a nucleic acid encoding an antibody thatspecifically binds REMODELIN, and the like. In one aspect, therecombinant cell can be transiently transfected with a vector (e.g., aplasmid, and the like) encoding a portion of the nucleic acid encodingREMODELIN. The nucleic acid need not be integrated into the cell genomenor does it need to be expressed in the cell. Moreover, the cell may bea prokaryotic or a eukaryotic cell and the invention should not beconstrued to be limited to any particular cell line or cell type. Suchcells include, but are not limited to, fibroblasts, mouse stem cells,amphibian oocytes, osteoblasts, smooth muscle cells, endothelial cells,and the like.

[0340] In one aspect, the recombinant cell comprising an isolatednucleic acid encoding mammalian REMODELIN is used to produce atransgenic non-human mammal. That is, the exogenous nucleic acid, or“transgene” as it is also referred to herein, of the invention isintroduced into a cell, and the cell is then used to generate thenon-human transgenic mammal. The cell into which the transgene isintroduced is preferably an embryonic stem (ES) cell. However, theinvention should not be construed to be limited solely to ES cellscomprising the transgene of the invention nor to cells used to producetransgenic animals. Rather, a transgenic cell of the invention includes,but is not limited to, any cell derived from a transgenic animalcomprising a transgene, a cell comprising the transgene derived from achimeric animal derived from the transgenic ES cell, and any othercomprising the transgene which may or may not be used to generate anon-human transgenic mammal.

[0341] Further, it is important to note that the purpose oftransgene-comprising, i.e., recombinant, cells should not be construedto be limited to the generation of transgenic mammals. Rather, theinvention should be construed to include any cell type into which anucleic acid encoding a mammalian REMODELIN is introduced, including,without limitation, a prokaryotic cell and a eukaryotic cell comprisingan isolated nucleic acid encoding mammalian REMODELIN.

[0342] When the cell is a eukaryotic cell, the cell may be anyeukaryotic cell which, when the transgene of the invention is introducedtherein, and the protein encoded by the desired gene is no longerexpressed therefrom, a benefit is obtained. Such a benefit may includethe fact that there has been provided a system in which lack ofexpression of the desired gene can be studied in vitro in the laboratoryor in a mammal in which the cell resides, a system wherein cellscomprising the introduced gene deletion can be used as research,diagnostic and therapeutic tools, and a system wherein animal models aregenerated which are useful for the development of new diagnostic andtherapeutic tools for selected disease states in a mammal including, forexample, negative remodeling, arterial restenosis, and the like. Thatis, one skilled in the art would appreciate, based upon the disclosureprovided herein, that because proliferation of fibroblasts with scartissue formation is part of any would healing process, selected diseasestates or processes associated with such proliferation that can beinvestigated by assessing REMODELIN expression include, but are notlimited to, wound healing, bone formation, bone fracture healing, andfibrosis of any organ.

[0343] Alternatively, the invention includes a eukaryotic cell which,when the transgene of the invention is introduced therein, and theprotein encoded by the desired gene is expressed therefrom where it wasnot previously present or expressed in the cell or where it is nowexpressed at a level or under circumstances different than that beforethe transgene was introduced, a benefit is obtained. Such a benefit mayinclude the fact that there has been provided a system in the expressionof the desired gene can be studied in vitro in the laboratory or in amammal in which the cell resides, a system wherein cells comprising theintroduced gene can be used as research, diagnostic and therapeutictools, and a system wherein animal models are generated which are usefulfor the development of new diagnostic and therapeutic tools for selecteddisease states in a mammal.

[0344] Such cell expressing an isolated nucleic acid encoding REMODELINcan be used to provide REMODELIN to a cell, tissue, or whole animalwhere a higher level of REMODELIN can be useful to treat or alleviate adisease, disorder or condition associated with low level of REMODELINexpression and/or activity. Such diseases, disorders or conditions caninclude, but are not limited to, wound healing, bone formation, and bonefracture healing, and the like. Moreover, one skilled in the art wouldunderstand that one goal of a wound healing response is to regainmechanical strength and structural support. REMODELIN is expressedduring such a healing response. Additional expression of REMODELIN couldthus lead to accelerated wound healing, bone growth, and fracturehealing. Therefore, the invention includes a cell expressing REMODELINto increase or induce REMODELIN expression, translation, and/oractivity, where increasing REMODELIN expression, protein level, and/oractivity can be useful to treat or alleviate a disease, disorder orcondition.

[0345] One of ordinary skill would appreciate, based upon the disclosureprovided herein, that a “knock-in” or “knock-out” vector of theinvention comprises at least two sequences homologous to two portions ofthe nucleic acid which is to be replaced or deleted, respectively. Thetwo sequences are homologous with sequences that flank the gene; thatis, one sequence is homologous with a region at or near the 5′ portionof the coding sequence of the nucleic acid encoding REMODELIN and theother sequence is further downstream from the first. One skilled in theart would appreciate, based upon the disclosure provided herein, thatthe present invention is not limited to any specific flanking nucleicacid sequences. Instead, the targeting vector may comprise two sequenceswhich remove some or all (i.e., a “knock-out” vector) or which insert(i.e., a “knock-in” vector) a nucleic acid encoding REMODELIN, or afragment thereof, from or into a mammalian genome, respectively. Thecrucial feature of the targeting vector is that it comprise sufficientportions of two sequences located towards opposite, i.e., 5′ and 3′,ends of the REMODELIN open reading frame (ORF) in the case of a“knock-out” vector, to allow deletion/insertion by homologousrecombination to occur such that all or a portion of the nucleic acidencoding REMODELIN is deleted from or inserted into a location on amammalian chromosome.

[0346] The design of transgenes and knock-in and knock-out targetingvectors is well-known in the art and is described in standard treatisessuch as Sambrook et al. (1989, Molecular Cloning: A Laboratory Manual,Cold Spring Harbor Laboratory, New York), and in Ausubel et al. (1997,Current Protocols in Molecular Biology, John Wiley & Sons, New York),and the like. The upstream and downstream portions flanking or withinthe REMODELIN coding region to be used in the targeting vector may beeasily selected based upon known methods and following the teachingsdisclosed herein based on the disclosure provided herein including thenucleic and amino acid sequences of both rat and human REMODELIN. Armedwith these sequences, one of ordinary skill in the art would be able toconstruct the transgenes and knock-out vectors of the invention.

[0347] The invention further includes a knock-out targeting vectorcomprising a nucleic acid encoding a selectable marker such as, forexample, a nucleic acid encoding the neo^(R) gene thereby allowing theselection of transgenic a cell where the nucleic acid encodingREMODELIN, or a portion thereof, has been deleted and replaced with theneomycin resistance gene by the cell's ability to grow in the presenceof G418. However, the present invention should not be construed to belimited to neomycin resistance as a selectable marker. Rather, otherselectable markers well-known in the art may be used in the knock-outtargeting vector to allow selection of recombinant cells where theREMODELIN gene has been deleted and/or inactivated and replaced by thenucleic acid encoding the selectable marker of choice. Methods ofselecting and incorporating a selectable marker into a vector arewell-known in the art and are describe in, for example, Sambrook et al.(1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), and in Ausubel et al. (1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York).

[0348] As noted herein, the invention includes a non-human transgenicmammal comprising an exogenous nucleic acid inserted into a desired sitein the genome thereof thereby deleting the coding region of a desiredendogenous target gene, i.e., a knock-out transgenic mammal. Further,the invention includes a transgenic non-human mammal wherein anexogenous nucleic acid encoding REMODELIN is inserted into a site thegenome, i.e., a “knock-in” transgenic mammal. The knock-in transgeneinserted may comprise various nucleic acids encoding, for example, a tagpolypeptide, a promoter/regulatory region operably linked to the nucleicacid encoding REMODELIN not normally present in the cell or nottypically operably linked to REMODELIN.

[0349] The generation of the non-human transgenic mammal of theinvention is preferably accomplished using the method which is nowdescribed. However, the invention should in no way be construed as beinglimited solely to the use of this method, in that, other methods can beused to generate the desired knock-out mammal.

[0350] In the preferred method of generating a non-human transgenicmammal, ES cells are generated comprising the transgene of the inventionand the cells are then used to generate the knock-out animal essentiallyas described in Nagy and Rossant (1993, In: Gene Targeting, A PracticalApproach, pp.146-179, Joyner ed., IRL Press). ES cells behave as normalembryonic cells if they are returned to the embryonic environment byinjection into a host blastocyst or aggregate with blastomere stageembryos. When so returned, the cells have the full potential to developalong all lineages of the embryo. Thus, it is possible, to obtain EScells, introduce a desired DNA therein, and then return the cell to theembryonic environment for development into mature mammalian cells,wherein the desired DNA may be expressed.

[0351] Precise protocols for the generation of transgenic mice aredisclosed in Nagy and Rossant (1993, In: Gene Targeting, A PracticalApproach, Joyner ed. IRL Press, pp. 146-179). and are therefore notrepeated herein. Transfection or transduction of ES cells in order tointroduce the desired DNA therein is accomplished using standardprotocols, such as those described, for example, in Sambrook et al.(1989, Molecular Cloning: A Laboratory Manual, Cold Spring HarborLaboratory, New York), and in Ausubel et al. (1997, Current Protocols inMolecular Biology, John Wiley & Sons, New York). Preferably, the desiredDNA contained within the transgene of the invention is electroporatedinto ES cells, and the cells are propagated as described in Soriano etal. (1991, Cell 64:693-702).

[0352] Introduction of an isolated nucleic acid into the fertilized eggof the mammal is accomplished by any number of standard techniques intransgenic technology (Hogan et al., 1986, Manipulating the MouseEmbryo: A Laboratory Manual, Cold Spring Harbor, N.Y.). Most commonly,the nucleic acid is introduced into the embryo by way of microinjection.

[0353] Once the nucleic acid is introduced into the egg, the egg isincubated for a short period of time and is then transferred into apseudopregnant mammal of the same species from which the egg wasobtained as described, for example, in Hogan et al. (1986, Manipulatingthe Mouse Embryo: A Laboratory Manual, Cold Spring Harbor, N.Y.).Typically, many eggs are injected per experiment, and approximatelytwo-thirds of the eggs survive the procedure. About twenty viable eggsare then transferred into pseudopregnant animals, and usually four toten of the viable eggs so transferred will develop into live pups.

[0354] Any mammalian REMODELIN gene may be used in the methods describedherein to produce a transgenic mammal or a transgenic cell harboring atransgene comprising a deletion of all or part of that mammalianREMODELIN gene. Preferably, a rodent REMODELIN gene such as, e.g., ratREMODELIN (SEQ ID NO: 1), encoding rat REMODELIN_(S) (SEQ ID NO: 2) andrat REMODELIN_(L) (SEQ ID NO: 5), is used, and human REMODELIN (SEQ IDNO: 3) gene, is also used.

[0355] The transgenic mammal of the invention can be any species ofmammal. Thus, the invention should be construed to include generation oftransgenic mammals encoding the chimeric nucleic acid, which mammalsinclude mice, hamsters, rats, rabbits, pigs, sheep and cattle. Themethods described herein for generation of transgenic mice can beanalogously applied using any mammalian species. Preferably, thetransgenic mammal of the invention is a rodent and even more preferably,the transgenic mammal of the invention is a mouse. By way of example,Lukkarinen et al. (1997, Stroke 28:639-645), teaches that geneconstructs which enable the generation of transgenic mice also enablethe generation of other transgenic rodents, including rats. Similarly,nullizygous mutations in a genetic locus of an animal of one species canbe replicated in an animal of another species having a genetic locushighly homologous to the first species.

[0356] To identify the transgenic mammals of the invention, pups areexamined for the presence of the isolated nucleic acid using standardtechnology such as Southern blot hybridization, PCR, and/or RT-PCR.Expression of the nucleic acid in the cells and in the tissues of themammal is also assessed using ordinary technology described herein.Further, the presence or absence of REMODELIN in the circulating bloodof the transgenic animal can be determined, if the protein is secreted,by using, -for example, Western blot analysis, or using standard methodsfor protein detection that are well-known in the art.

[0357] Cells obtained from the transgenic mammal of the invention, whichare also considered “transgenic cells” as the term is used herein,encompass such as cells as those obtained from the REMODELIN (+/−) and(−/−) transgenic non-human mammal described elsewhere herein, are usefulsystems for modeling diseases and symptoms of mammals which are believedto be associated with altered levels of REMODELIN expression such asnegative remodeling, arterial restenosis, adventitial fibrosis, woundhealing, bone formation, bone density, dorsal closure, spina bifida-likeconditions, and any other disease, disorder or condition associated withan altered level of REMODELIN expression. Moreover, as a marker of apathway(s) associated with cell proliferation and cell migration,REMODELIN expression levels are also useful indicators in assessment ofvarious diseases, disorders or conditions associated with excessive orimpaired wound healing (e.g., skin wound healing) and conditionsassociated with excessive or impaired bone formation, and the like.

[0358] Particularly suitable are cells derived from a tissue of thenon-human knock-out or knock-in transgenic mammal described herein,wherein the transgene comprising the REMODELIN gene is expressed orinhibits expression of REMODELIN in various tissues. By way of example,cell types from which such cells are derived include fibroblasts andlike cells of (1) the REMODELIN (+/+), (+/−) and (−/−) non-humantransgenic liveborn mammal, (2) the REMODELIN (+/+), (−/−) or (+/−)fetal animal, and (3) placental cell lines obtained from the REMODELIN(+/+), (−/−) and (+/−) fetus and liveborn mammal.

[0359] One skilled in the art would appreciate, based upon thisdisclosure, that cells comprising decreased levels of REMODELIN protein,decreased level of REMODELIN activity, or both, include, but are notlimited to, cells expressing inhibitors of REMODELIN expression (e.g.,antisense or ribozyme molecules).

[0360] Methods and compositions useful for maintaining mammalian cellsin culture are well known in the art, wherein the mammalian cells areobtained from a mammal including, but not limited to, cells obtainedfrom a mouse such as the transgenic mouse described herein.

[0361] The recombinant cell of the invention can be used to study theeffect of qualitative and quantitative alterations in REMODELIN levelson cell signal transduction systems. This is because the fact that thedata disclosed herein indicate that REMODELIN is involved in TGF-βsignaling pathways. Further, the recombinant cell can be used to produceREMODELIN for use for therapeutic and/or diagnostic purposes. That is, arecombinant cell expressing REMODELIN can be used to produce largeamounts of purified and isolated REMODELIN that can be administered totreat or alleviate a disease, disorder or condition associated with orcaused by a decreased level of REMODELIN.

[0362] Alternatively, recombinant cells expressing REMODELIN can beadministered in ex vivo and in vivo therapies where administering therecombinant cells thereby administers the protein to a cell, a tissue,and/or an animal. Additionally, the recombinant cells are useful for thediscovery of REMODELIN ligand(s) and REMODELIN signaling pathway(s).

[0363] The recombinant cell of the invention may be used to study theeffects of elevated or decreased REMODELIN levels on cell homeostasisand cell proliferation and/or migration since REMODELIN has beenhypothesized to play a role in cell migration, adventitial fibrosis,arterial restenosis, negative remodeling, and the like

[0364] The recombinant cell of the invention, wherein the cell has beenengineered such that it does not express REMODELIN, or expresses reducedor altered REMODELIN lacking biological activity, can also be used in exvivo and in vivo cell therapies where either an animal's own cells(e.g., fibroblasts, and the like), or those of a syngeneic matcheddonor, are recombinantly engineered as described elsewhere herein (e.g.,by insertion of an antisense nucleic acid or a knock-out vector suchthat REMODELIN expression and/or protein levels are thereby reduced inthe recombinant cell), and the recombinant cell is administered to therecipient animal. In this way, recombinant cells that express REMODELINat a reduced level can be administered to an animal whose own cellsexpress increased levels of REMODELIN thereby treating or alleviating adisease, disorder or condition associated with or mediated by increasedREMODELIN expression as disclosed elsewhere herein.

[0365] The transgenic mammal of the invention, rendered susceptible toadventitial fibrosis, arterial restenosis, and the like, such as, forexample, a REMODELIN knock-out mouse, can be used to study thepathogenesis of these diseases and the potential role of REMODELINtherein.

[0366] Further, the transgenic mammal and/or cell of the invention maybe used to further study the subcellular localization of REMODELIN.

[0367] Also, the transgenic mammal (both +/− and −/− live born andfetuses) and/or cell of the invention may be used to study to role(s) ofREMODELIN in cell migration and proliferation, and TGF-β signaling toelucidate the target(s) of REMODELIN action as well as any receptor(s)and/or ligands that bind with REMODELIN to mediate its effect(s) in thecell.

[0368] VI. Antibodies

[0369] The invention also includes an antibody that specifically bindsREMODELIN, or a fragment thereof.

[0370] One skilled in the art would understand, based upon thedisclosure provided herein, that an antibody that specifically bindsREMODELIN, binds with a protein of the invention, such as, but notlimited to rat REMODELIN_(S), human REMODELIN, and rat REMODELIN_(L), oran immunogenic portion thereof. In one embodiment, the antibody isdirected to rat REMODELIN comprising the amino acid sequence of SEQ IDNO: 2 and SEQ ID NO: 5, and an antibody directed to human REMODELIN,comprising the amino acid sequence SEQ ID NO: 4.

[0371] Polyclonal antibodies are generated by immunizing rabbitsaccording to standard immunological techniques well-known in the art(see, e.g., Harlow et al., 1988, In: Antibodies, A Laboratory Manual,Cold Spring Harbor, N.Y.). Such techniques include immunizing an animalwith a chimeric protein comprising a portion of another protein such asa maltose binding protein or glutathione (GSH) tag polypeptide portion,and/or a moiety such that the REMODELIN portion is rendered immunogenic(e.g., REMODELIN conjugated with keyhole limpet hemocyanin, KLH) and aportion comprising the respective rodent and/or human REMODELIN aminoacid residues. The chimeric proteins are produced by cloning theappropriate nucleic acids encoding REMODELIN (e.g., SEQ ID NO: 1 and SEQID NO: 3) into a plasmid vector suitable for this purpose, such as butnot limited to, pMAL-2 or pCMX.

[0372] However, the invention should not be construed as being limitedsolely to these antibodies or to these portions of the protein antigens.Rather, the invention should be construed to include other antibodies,as that term is defined elsewhere herein, to rat and human REMODELIN, orportions thereof. Further, the present invention should be construed toencompass antibodies, inter alia, bind with REMODELIN and they are ableto bind REMODELIN present on Western blots, in immunohistochemicalstaining of tissues thereby localizing REMODELIN in the tissues, and inimmunofluorescence microscopy of a cell transiently transfected with anucleic acid encoding at least a portion of REMODELIN.

[0373] One skilled in the art would appreciate, based upon thedisclosure provided herein, that the antibody can specifically bind withany portion of the protein and the full-length protein can be used togenerate antibodies specific therefor. However, the present invention isnot limited to using the full-length protein as an immunogen. Rather,the present invention includes using an immunogenic portion of theprotein to produce an antibody that specifically binds with mammalianREMODELIN. That is, the invention includes immunizing an animal using animmunogenic portion, or antigenic determinant, of the REMODELIN protein.Such immunogenic portions can include, but are not limited to, thecarboxy-terminal 15 amino acids (GWNSVSRIIIEELPK) (SEQ ID NO: 7). Theantibodies can be produced by immunizing an animal such as, but notlimited to, a rabbit or a mouse, with a protein of the invention, or aportion thereof, or by immunizing an animal using a protein comprisingat least a portion of REMODELIN, or a fusion protein including a tagpolypeptide portion comprising, for example, a maltose binding proteintag polypeptide portion covalently linked with a portion comprising theappropriate REMODELIN amino acid residues. One skilled in the art wouldappreciate, based upon the disclosure provided herein, that smallerfragments of these proteins can also be used to produce antibodies thatspecifically bind REMODELIN.

[0374] One skilled in the art would appreciate, based upon thedisclosure provided herein, that various portions of an isolatedREMODELIN polypeptide can be used to generate antibodies to eitherhighly conserved regions of REMODELIN or to non-conserved regions of thepolypeptide. As disclosed elsewhere herein, REMODELIN comprises variousconserved domains including, but not limited to, a putative signalpeptide from about amino acid residue 1 to about amino acid residue 32transmembrane domain/signal peptide (amino acid residues from about 1 to32); a CK2 phosphorylation domain (amino acid residues from about 31 to34); an N-myristoylation domain (amino acid residues from about 69 to74); a CK2 phosphorylation domain (amino acid residues from about 99 to102); an N-myristoylation domain (amino acid residues from about 119 to124); a PKC phosphorylation domain (amino acid residues from about 146to 148); an N-myristoylation domain (amino acid residues from about 165to 170); an N-glycosylation domain (amino acid residues from about 188to 191); a CK2 phosphorylation domain (amino acid residues from about197 to 200); an N-myristoylation domain (amino acid residues from about201 to 206); an N-myristoylation domain (amino acid residues from about205 to 210); and a CK2 phosphorylation domain (amino acid residues fromabout 219 to 222). These domains are also present in rat and humanREMODELINs (see, e.g., SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5).

[0375] Once armed with the sequence of REMODELIN and the detailedanalysis localizing the various conserved and non-conserved domains ofthe protein, the skilled artisan would understand, based upon thedisclosure provided herein, how to obtain antibodies specific for thevarious portions of a mammalian REMODELIN polypeptide using methodswell-known in the art or to be developed, as well as methods disclosedherein.

[0376] Further, the skilled artisan, based upon the disclosure providedherein, would appreciate that the non-conserved regions of a protein ofinterest can be more immunogenic than the highly conserved regions whichare conserved among various organisms. Further, immunization using anon-conserved immunogenic portion can produce antibodies specific forthe non-conserved region thereby producing antibodies that do notcross-react with other proteins which can share one or more conservedportions. Thus, one skilled in the art would appreciate, based upon thedisclosure provided herein, that the non-conserved regions of eachREMODELIN molecule can be used to produce antibodies that are specificonly for that REMODELIN and do not cross-react non-specifically withother REMODELINs or with other proteins.

[0377] Alternatively, the skilled artisan would also understand, basedupon the disclosure provided herein, that antibodies developed using aregion that is conserved among one or more REMODELIN molecule can beused to produce antibodies that react specifically with one or moreREMODELIN molecule. Methods for producing antibodies that specificallybind with a conserved protein domain which may otherwise be lessimmunogenic than other portions of the protein are well-known in the artand include, but are not limited to, conjugating the protein fragment ofinterest to a molecule (e.g., keyhole limpet hemocyanin, and the like),thereby rendering the protein domain immunogenic, or by the use ofadjuvants (e.g., Freund's complete and/or incomplete adjuvant, and thelike), or both. Thus, the invention encompasses antibodies thatrecognize at least one REMODELIN and antibodies that specifically bindwith more than one REMODELIN, including antibodies that specificallybind with all REMODELIN.

[0378] One skilled in the art would appreciate, based upon thedisclosure provided herein, which portions of REMODELIN are lesshomologous with other proteins sharing conserved domains. However, thepresent invention is not limited to any particular domain; instead, theskilled artisan would understand that other non-conserved regions of theREMODELIN proteins of the invention can be used to produce theantibodies of the invention as disclosed herein.

[0379] Therefore, the skilled artisan would appreciate, based upon thedisclosure provided herein, that the present invention encompassesantibodies that neutralize and/or inhibit REMODELIN activity (e.g., byinhibiting necessary REMODELIN receptor/ligand interactions) whichantibodies can recognize one or more REMODELINs, including, but notlimited to, rat REMODELIN_(S), rat REMODELIN_(L), and human REMODELIN,as well as REMODELINs from various species (e.g., mouse REMODELIN).

[0380] The invention should not be construed as being limited solely tothe antibodies disclosed herein or to any particular immunogenic portionof the proteins of the invention. Rather, the invention should beconstrued to include other antibodies, as that term is defined elsewhereherein, to REMODELIN, or portions thereof, or to proteins sharing atleast about 6% homology with a polypeptide having the amino acidsequence of at least one of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:5. Preferably, the polypeptide is about 10% homologous, more preferably,at least about 15% homologous, more preferably, at least about 20%homologous, even more preferably, at least about 25% homologous, morepreferably, at least about 30% homologous, preferably, at least about35% homologous, even more preferably, at least about 40% homologous,even more preferably, at least about 45% homologous, yet morepreferably, at least about 50% homologous, more preferably, at leastabout 55% homologous, more preferably, at least about 60% homologous,even more preferably, at least about 65% homologous, yet morepreferably, at least about 70% homologous, more preferably, at leastabout 75% homologous, even more preferably, at least about 80%homologous, yet more preferably, at least about 85% homologous, morepreferably, at least about 90% homologous, even more preferably, atleast about 95% homologous, and most preferably, at least about 99%homologous to at least one of rat REMODELIN_(S) (SEQ ID NO: 2), humanREMODELIN (SEQ ID NO: 4), and rat REMODELIN_(L) (SEQ ID NO: 5). Morepreferably, the polypeptide that specifically binds with an antibodyspecific for mammalian REMODELIN is at least one of rat REMODELIN_(S)(SEQ ID NO: 2), human REMODELIN (SEQ ID NO: 4), and rat REMODELIN_(L)(SEQ ID NO: 5). Most preferably, the polypeptide that specifically bindswith an antibody that specifically binds with a mammalian REMODELIN isat least one of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO: 5.

[0381] Further, the skilled artisan would appreciate, based upon thedisclosure provided herein, that amino acid sequences that may elicitantibodies that non-specifically cross-react with a non-REMODELINprotein can also be excluded from use as immunogens. For example, suchamino acid sequences include, but are not limited to, an amino acidsequence comprising collagen alpha-2 (IV) chain precursor (GenBank Acc.No. P27393), which shares about 62% identity with REMODELIN over a 35amino acid stretch. Thus, such a portion sharing at least about 62%identity over 35 amino acids of REMODELIN would not be used to producethe antibodies of the invention.

[0382] The invention encompasses polyclonal, monoclonal, syntheticantibodies, and the like. One skilled in the art would understand, basedupon the disclosure provided herein, that the crucial feature of theantibody of the invention is that the antibody bind specifically withREMODELIN. That is, the antibody of the invention recognizes REMODELIN,or a fragment thereof (e.g., an immunogenic portion or antigenicdeterminant thereof), as demonstrated by antibody binding REMODELIN onWestern blots, in immunostaining of cells, and/o immunoprecipitation ofREMODELIN, using standard methods well-known in the art.

[0383] One skilled in the art would appreciate, based upon thedisclosure provided herein, that the antibodies can be used to localizethe relevant protein in a cell and to study the role(s) of the antigenrecognized thereby in cell processes. Moreover, the antibodies can beused to detect and or measure the amount of protein present in abiological sample using well-known methods such as, but not limited to,Western blotting and enzyme-linked immunosorbent assay (ELISA).Moreover, the antibodies can be used to immunoprecipitate and/orimmuno-affinity purify their cognate antigen using methods well-known inthe art.

[0384] In addition, the antibody can be used to decrease the level ofREMODELIN in a cell thereby inhibiting the effect(s) of REMODELIN in acell. Thus, by administering the antibody to a cell or to the tissues ofan animal or to the animal itself, the required REMODELINreceptor/ligand interactions are therefore inhibited such that theeffect of REMODELIN-mediated signaling are also inhibited. One skilledin the art would understand, based upon the disclosure provided herein,that detectable effects upon inhibiting REMODELIN ligand/receptorinteraction using an anti-REMODELIN antibody can include, but are notlimited to, decreased proliferation of cells, decreased cell migration,decreased negative modeling, decreased adventitial fibrosis, decreasedarterial restenosis, decreased fibrosis in any organ or tissue,decreased ossification or bone formation, and the like.

[0385] The generation of polyclonal antibodies is accomplished byinoculating the desired animal with the antigen and isolating antibodieswhich specifically bind the antigen therefrom using standard antibodyproduction methods such as those described in, for example, Harlow etal. (1988, In: Antibodies, A Laboratory Manual, Cold Spring Harbor,N.Y.).

[0386] Monoclonal antibodies directed against full length or peptidefragments of a protein or peptide may be prepared using any well knownmonoclonal antibody preparation procedures, such as those described, forexample, in Harlow et al. (1988, In: Antibodies, A Laboratory Manual,Cold Spring Harbor, N.Y.) and in Tuszynski et al. (1988, Blood,72:109-115). Quantities of the desired peptide may also be synthesizedusing chemical synthesis technology. Alternatively, DNA encoding thedesired peptide may be cloned and expressed from an appropriate promotersequence in cells suitable for the generation of large quantities ofpeptide. Monoclonal antibodies directed against the peptide aregenerated from mice immunized with the peptide using standard proceduresas referenced herein.

[0387] Nucleic acid encoding the monoclonal antibody obtained using theprocedures described herein may be cloned and sequenced using technologywhich is available in the art, and is described, for example, in Wrightet al. (1992, Critical Rev. Immunol. 12:125-168), and the referencescited therein.

[0388] Further, the antibody of the invention may be “humanized” usingthe technology described in, for example, Wright et al. (supra), and inthe references cited therein, and in Gu et al. (1997, Thrombosis andHematocyst 77:755-759), and other methods of humanizing antibodieswell-known in the art or to be developed.

[0389] To generate a phage antibody library, a cDNA library is firstobtained from mRNA which is isolated from cells, e.g., the hybridoma,which express the desired protein to be expressed on the phage surface,e.g., the desired antibody. cDNA copies of the mRNA are produced usingreverse transcriptase. cDNA which specifies immunoglobulin fragments areobtained by PCR and the resulting DNA is cloned into a suitablebacteriophage vector to generate a bacteriophage DNA library comprisingDNA specifying immunoglobulin genes. The procedures for making abacteriophage library comprising heterologous DNA are well known in theart and are described, for example, in Sambrook et al., supra.

[0390] Bacteriophage which encode the desired antibody, may beengineered such that the protein is displayed on the surface thereof insuch a manner that it is available for binding to its correspondingbinding protein, e.g., the antigen against which the antibody isdirected. Thus, when bacteriophage which express a specific antibody areincubated in the presence of a cell which expresses the correspondingantigen, the bacteriophage will bind to the cell. Bacteriophage which donot express the antibody will not bind to the cell. Such panningtechniques are well known in the art and are described for example, inWright et al. (supra).

[0391] Processes such as those described above, have been developed forthe production of human antibodies using M13 bacteriophage display(Burton et al., 1994, Adv. Immunol. 57:191-280). Essentially, a cDNAlibrary is generated from mRNA obtained from a population ofantibody-producing cells. The mRNA encodes rearranged immunoglobulingenes and thus, the cDNA encodes the same. Amplified cDNA is cloned intoM13 expression vectors creating a library of phage which express humanFab fragments on their surface. Phage which display the antibody ofinterest are selected by antigen binding and are propagated in bacteriato produce soluble human Fab immunoglobulin. Thus, in contrast toconventional monoclonal antibody synthesis, this procedure immortalizesDNA encoding human immunoglobulin rather than cells which express humanimmunoglobulin.

[0392] The procedures just presented describe the generation of phagewhich encode the Fab portion of an antibody molecule. However, theinvention should not be construed to be limited solely to the generationof phage encoding Fab antibodies. Rather, phage which encode singlechain antibodies (scFv/phage antibody libraries) are also included inthe invention. Fab molecules comprise the entire Ig light chain, thatis, they comprise both the variable and constant region of the lightchain, but include only the variable region and first constant regiondomain (CH1) of the heavy chain. Single chain antibody moleculescomprise a single chain of protein comprising the Ig Fv fragment. An IgFv fragment includes only the variable regions of the heavy and lightchains of the antibody, having no constant region contained therein.Phage libraries comprising scFv DNA may be generated following theprocedures described in Marks et al. (1991, J. Mol. Biol. 222:581-597).Panning of phage so generated for the isolation of a desired antibody isconducted in a manner similar to that described for phage librariescomprising Fab DNA.

[0393] The invention should also be construed to include synthetic phagedisplay libraries in which the heavy and light chain variable regionsmay be synthesized such that they include nearly all possiblespecificities (Barbas, 1995, Nature Medicine 1:837-839; de Kruif et al.1995, J. Mol. Biol. 248:97-105).

[0394] VII. Compositions

[0395] The invention includes a composition comprising an isolatednucleic complementary to a nucleic acid, or a portion thereof, encodinga mammalian REMODELIN, which is in an antisense orientation with respectto transcription. Preferably, the composition comprises apharmaceutically acceptable carrier.

[0396] The invention includes a composition comprising an isolatedmammalian REMODELIN polypeptide as described herein. Preferably, thecomposition comprises a pharmaceutically-acceptable carrier.

[0397] The invention also includes a composition comprising an antibodythat specifically binds REMODELIN. Preferably, the composition comprisesa pharmaceutically-acceptable carrier.

[0398] The invention further includes a composition comprising anisolated nucleic acid encoding a mammalian REMODELIN. Preferably, thecomposition comprises a pharmaceutically acceptable carrier.

[0399] The compositions can be used to administer REMODELIN to a cell, atissue, or an animal or to inhibit expression of REMODELIN in a cell, atissue, or an animal. The compositions are useful to treat a disease,disorder or condition mediated by altered expression of REMODELIN suchthat decreasing or increasing REMODELIN expression or the level of theprotein in a cell, tissue, or animal, is beneficial to the animal. Thatis, where a disease, disorder or condition in an animal is mediated byor associate with altered level of REMODELIN expression or proteinlevel, the composition can be used to modulate such expression orprotein level of REMODELIN.

[0400] For administration to the mammal, a polypeptide, or a nucleicacid encoding it, and/or an antisense nucleic acid complementary to allor a portion thereof, can be suspended in any pharmaceuticallyacceptable carrier, for example, HEPES buffered saline at a pH of about7.8.

[0401] Other pharmaceutically acceptable carriers which are usefulinclude, but are not limited to, glycerol, water, saline, ethanol andother pharmaceutically acceptable salt solutions such as phosphates andsalts of organic acids. Examples of these and other pharmaceuticallyacceptable carriers are described in Remington's Pharmaceutical Sciences(1991, Mack Publication Co., New Jersey).

[0402] The pharmaceutical compositions may be prepared, packaged, orsold in the form of a sterile injectable aqueous or oily suspension orsolution. This suspension or solution may be formulated according to theknown art, and may comprise, in addition to the active ingredient,additional ingredients such as the dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butane diol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides.

[0403] Pharmaceutical compositions that are useful in the methods of theinvention may be administered, prepared, packaged, and/or sold informulations suitable for oral, rectal, vaginal, parenteral, topical,pulmonary, intranasal, buccal, ophthalmic, or another route ofadministration. Other contemplated formulations include projectednanoparticles, liposomal preparations, resealed erythrocytes containingthe active ingredient, and immunologically-based formulations.

[0404] The compositions of the invention may be administered vianumerous routes, including, but not limited to, oral, rectal, vaginal,parenteral, topical, pulmonary, intranasal, buccal, or ophthalmicadministration routes. The route(s) of administration will be readilyapparent to the skilled artisan and will depend upon any number offactors including the type and severity of the disease being treated,the type and age of the veterinary or human patient being treated, andthe like.

[0405] Pharmaceutical compositions that are useful in the methods of theinvention may be administered systemically in oral solid formulations,ophthalmic, suppository, aerosol, topical or other similar formulations.In addition to the compound such as heparan sulfate, or a biologicalequivalent thereof, such pharmaceutical compositions may containpharmaceutically-acceptable carriers and other ingredients known toenhance and facilitate drug administration. Other possible formulations,such as nanoparticles, liposomes, resealed erythrocytes, andimmunologically based systems may also be used to administer REMODELINand/or a nucleic acid encoding the same according to the methods of theinvention.

[0406] Compounds which are identified using any of the methods describedherein may be formulated and administered to a mammal for treatment ofarterial restenosis, adventitial fibrosis, fibrosis in any organ ortissue, negative remodeling, excessive bone formation, excessiveossification, and the like, are now described.

[0407] The invention encompasses the preparation and use ofpharmaceutical compositions comprising a compound useful for treatmentof arterial restenosis, adventitial fibrosis, negative remodeling, andthe like, as an active ingredient. Such a pharmaceutical composition mayconsist of the active ingredient alone, in a form suitable foradministration to a subject, or the pharmaceutical composition maycomprise the active ingredient and one or more pharmaceuticallyacceptable carriers, one or more additional ingredients, or somecombination of these. The active ingredient may be present in thepharmaceutical composition in the form of a physiologically acceptableester or salt, such as in combination with a physiologically acceptablecation or anion, as is well known in the art.

[0408] As used herein, the term “pharmaceutically acceptable carrier”means a chemical composition with which the active ingredient may becombined and which, following the combination, can be used to administerthe active ingredient to a subject.

[0409] As used herein, the term “physiologically acceptable” ester orsalt means an ester or salt form of the active ingredient which iscompatible with any other ingredients of the pharmaceutical composition,which is not deleterious to the subject to which the composition is tobe administered.

[0410] The formulations of the pharmaceutical compositions describedherein may be prepared by any method known or hereafter developed in theart of pharmacology. In general, such preparatory methods include thestep of bringing the active ingredient into association with a carrieror one or more other accessory ingredients, and then, if necessary ordesirable, shaping or packaging the product into a desired single- ormulti-dose unit.

[0411] Although the descriptions of pharmaceutical compositions providedherein are principally directed to pharmaceutical compositions which aresuitable for ethical administration to humans, it will be understood bythe skilled artisan that such compositions are generally suitable foradministration to animals of all sorts. Modification of pharmaceuticalcompositions suitable for administration to humans in order to renderthe compositions suitable for administration to various animals is wellunderstood, and the ordinarily skilled veterinary pharmacologist candesign and perform such modification with merely ordinary, if any,experimentation. Subjects to which administration of the pharmaceuticalcompositions of the invention is contemplated include, but are notlimited to, humans and other primates, mammals including commerciallyrelevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

[0412] Pharmaceutical compositions that are useful in the methods of theinvention may be prepared, packaged, or sold in formulations suitablefor oral, rectal, vaginal, parenteral, topical, pulmonary, intranasal,buccal, ophthalmic, intrathecal or another route of administration.Other contemplated formulations include projected nanoparticles,liposomal preparations, resealed erythrocytes containing the activeingredient, and immunologically-based formulations.

[0413] A pharmaceutical composition of the invention may be prepared,packaged, or sold in bulk, as a single unit dose, or as a plurality ofsingle unit doses. As used herein, a “unit dose” is discrete amount ofthe pharmaceutical composition comprising a predetermined amount of theactive ingredient. The amount of the active ingredient is generallyequal to the dosage of the active ingredient which would be administeredto a subject or a convenient fraction of such a dosage such as, forexample, one-half or one-third of such a dosage.

[0414] The relative amounts of the active ingredient, thepharmaceutically acceptable carrier, and any additional ingredients in apharmaceutical composition of the invention will vary, depending uponthe identity, size, and condition of the subject treated and furtherdepending upon the route by which the composition is to be administered.By way of example, the composition may comprise between 0.1% and 100%(w/w) active ingredient.

[0415] In addition to the active ingredient, a pharmaceuticalcomposition of the invention may further comprise one or more additionalpharmaceutically active agents. Particularly contemplated additionalagents include anti-emetics and scavengers such as cyanide and cyanatescavengers.

[0416] Controlled- or sustained-release formulations of a pharmaceuticalcomposition of the invention may be made using conventional technology.

[0417] A formulation of a pharmaceutical composition of the inventionsuitable for oral administration may be prepared, packaged, or sold inthe form of a discrete solid dose unit including, but not limited to, atablet, a hard or soft capsule, a cachet, a troche, or a lozenge, eachcontaining a predetermined amount of the active ingredient. Otherformulations suitable for oral administration include, but are notlimited to, a powdered or granular formulation, an aqueous or oilysuspension, an aqueous or oily solution, or an emulsion.

[0418] As used herein, an “oily” liquid is one which comprises acarbon-containing liquid molecule and which exhibits a less polarcharacter than water.

[0419] A tablet comprising the active ingredient may, for example, bemade by compressing or molding the active ingredient, optionally withone or more additional ingredients. Compressed tablets may be preparedby compressing, in a suitable device, the active ingredient in afree-flowing form such as a powder or granular preparation, optionallymixed with one or more of a binder, a lubricant, an excipient, a surfaceactive agent, and a dispersing agent. Molded tablets may be made bymolding, in a suitable device, a mixture of the active ingredient, apharmaceutically acceptable carrier, and at least sufficient liquid tomoisten the mixture. Pharmaceutically acceptable excipients used in themanufacture of tablets include, but are not limited to, inert diluents,granulating and disintegrating agents, binding agents, and lubricatingagents. Known dispersing agents include, but are not limited to, potatostarch and sodium starch glycollate. Known surface active agentsinclude, but are not limited to, sodium lauryl sulphate. Known diluentsinclude, but are not limited to, calcium carbonate, sodium carbonate,lactose, microcrystalline cellulose, calcium phosphate, calcium hydrogenphosphate, and sodium phosphate. Known granulating and disintegratingagents include, but are not limited to, corn starch and alginic acid.Known binding agents include, but are not limited to, gelatin, acacia,pre-gelatinized maize starch, polyvinylpyrrolidone, and hydroxypropylmethylcellulose. Known lubricating agents include, but are not limitedto, magnesium stearate, stearic acid, silica, and talc.

[0420] Tablets may be non-coated or they may be coated using knownmethods to achieve delayed disintegration in the gastrointestinal tractof a subject, thereby providing sustained release and absorption of theactive ingredient. By way of example, a material such as glycerylmonostearate or glyceryl distearate may be used to coat tablets. Furtherby way of example, tablets may be coated using methods described in U.S.Pat. Nos. 4,256,108; 4,160,452; and 4,265,874 to formosmotically-controlled release tablets. Tablets may further comprise asweetening agent, a flavoring agent, a coloring agent, a preservative,or some combination of these in order to provide pharmaceuticallyelegant and palatable preparation.

[0421] Hard capsules comprising the active ingredient may be made usinga physiologically degradable composition, such as gelatin. Such hardcapsules comprise the active ingredient, and may further compriseadditional ingredients including, for example, an inert solid diluentsuch as calcium carbonate, calcium phosphate, or kaolin.

[0422] Soft gelatin capsules comprising the active ingredient may bemade using a physiologically degradable composition, such as gelatin.Such soft capsules comprise the active ingredient, which may be mixedwith water or an oil medium such as peanut oil, liquid paraffin, orolive oil.

[0423] Liquid formulations of a pharmaceutical composition of theinvention which are suitable for oral administration may be prepared,packaged, and sold either in liquid form or in the form of a dry productintended for reconstitution with water or another suitable vehicle priorto use.

[0424] Liquid suspensions may be prepared using conventional methods toachieve suspension of the active ingredient in an aqueous or oilyvehicle. Aqueous vehicles include, for example, water and isotonicsaline. Oily vehicles include, for example, almond oil, oily esters,ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconutoil, fractionated vegetable oils, and mineral oils such as liquidparaffin. Liquid suspensions may further comprise one or more additionalingredients including, but not limited to, suspending agents, dispersingor wetting agents, emulsifying agents, demulcents, preservatives,buffers, salts, flavorings, coloring agents, and sweetening agents. Oilysuspensions may further comprise a thickening agent. Known suspendingagents include, but are not limited to, sorbitol syrup, hydrogenatededible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gumacacia, and cellulose derivatives such as sodium carboxymethylcellulose,methylcellulose, hydroxypropylmethylcellulose. Known dispersing orwetting agents include, but are not limited to, naturally-occurringphosphatides such as lecithin, condensation products of an alkyleneoxide with a fatty acid, with a long chain aliphatic alcohol, with apartial ester derived from a fatty acid and a hexitol, or with a partialester derived from a fatty acid and a hexitol anhydride (e.g.polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylenesorbitol monooleate, and polyoxyethylene sorbitan monooleate,respectively). Known emulsifying agents include, but are not limited to,lecithin and acacia. Known preservatives include, but are not limitedto, methyl, ethyl, or n-propyl-para-hydroxybenzoates, ascorbic acid, andsorbic acid. Known sweetening agents include, for example, glycerol,propylene glycol, sorbitol, sucrose, and saccharin. Known thickeningagents for oily suspensions include, for example, beeswax, hardparaffin, and cetyl alcohol.

[0425] Liquid solutions of the active ingredient in aqueous or oilysolvents may be prepared in substantially the same manner as liquidsuspensions, the primary difference being that the active ingredient isdissolved, rather than suspended in the solvent. Liquid solutions of thepharmaceutical composition of the invention may comprise each of thecomponents described with regard to liquid suspensions, it beingunderstood that suspending agents will not necessarily aid dissolutionof the active ingredient in the solvent. Aqueous solvents include, forexample, water and isotonic saline. Oily solvents include, for example,almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis,olive, sesame, or coconut oil, fractionated vegetable oils, and mineraloils such as liquid paraffin.

[0426] Powdered and granular formulations of a pharmaceuticalpreparation of the invention may be prepared using known methods. Suchformulations may be administered directly to a subject, used, forexample, to form tablets, to fill capsules, or to prepare an aqueous oroily suspension or solution by addition of an aqueous or oily vehiclethereto. Each of these formulations may further comprise one or more ofdispersing or wetting agent, a suspending agent, and a preservative.Additional excipients, such as fillers and sweetening, flavoring, orcoloring agents, may also be included in these formulations.

[0427] A pharmaceutical composition of the invention may also beprepared, packaged, or sold in the form of oil-in-water emulsion or awater-in-oil emulsion. The oily phase may be a vegetable oil such asolive or arachis oil, a mineral oil such as liquid paraffin, or acombination of these. Such compositions may further comprise one or moreemulsifying agents such as naturally occurring gums such as gum acaciaor gum tragacanth, naturally-occurring phosphatides such as soybean orlecithin phosphatide, esters or partial esters derived from combinationsof fatty acids and hexitol anhydrides such as sorbitan monooleate, andcondensation products of such partial esters with ethylene oxide such aspolyoxyethylene sorbitan monooleate. These emulsions may also containadditional ingredients including, for example, sweetening or flavoringagents.

[0428] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for rectal administration.Such a composition may be in the form of, for example, a suppository, aretention enema preparation, and a solution for rectal or colonicirrigation.

[0429] Suppository formulations may be made by combining the activeingredient with a non-irritating pharmaceutically acceptable excipientwhich is solid at ordinary room temperature (i.e., about 20° C.) andwhich is liquid at the rectal temperature of the subject (i.e., about37° C. in a healthy human). Suitable pharmaceutically acceptableexcipients include, but are not limited to, cocoa butter, polyethyleneglycols, and various glycerides. Suppository formulations may furthercomprise various additional ingredients including, but not limited to,antioxidants and preservatives.

[0430] Retention enema preparations or solutions for rectal or colonicirrigation may be made by combining the active ingredient with apharmaceutically acceptable liquid carrier. As is well known in the art,enema preparations may be administered using, and may be packagedwithin, a delivery device adapted to the rectal anatomy of the subject.Enema preparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants and preservatives.

[0431] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for vaginal administration.Such a composition may be in the form of, for example, a suppository, animpregnated or coated vaginally-insertable material such as a tampon, adouche preparation, or gel or cream or a solution for vaginalirrigation.

[0432] Methods for impregnating or coating a material with a chemicalcomposition are known in the art, and include, but are not limited tomethods of depositing or binding a chemical composition onto a surface,methods of incorporating a chemical composition into the structure of amaterial during the synthesis of the material (i.e., such as with aphysiologically degradable material), and methods of absorbing anaqueous or oily solution or suspension into an absorbent material, withor without subsequent drying.

[0433] Douche preparations or solutions for vaginal irrigation may bemade by combining the active ingredient with a pharmaceuticallyacceptable liquid carrier. As is well known in the art, douchepreparations may be administered using, and may be packaged within, adelivery device adapted to the vaginal anatomy of the subject. Douchepreparations may further comprise various additional ingredientsincluding, but not limited to, antioxidants, antibiotics, antifungalagents, and preservatives.

[0434] As used herein, “parenteral administration” of a pharmaceuticalcomposition includes any route of administration characterized byphysical breaching of a tissue of a subject and administration of thepharmaceutical composition through the breach in the tissue. Parenteraladministration thus includes, but is not limited to, administration of apharmaceutical composition by injection of the composition, byapplication of the composition through a surgical incision, byapplication of the composition through a tissue-penetrating non-surgicalwound, and the like. In particular, parenteral administration iscontemplated to include, but is not limited to, subcutaneous,intraperitoneal, intramuscular, intrastemal injection, and kidneydialytic infusion techniques.

[0435] Formulations of a pharmaceutical composition suitable forparenteral administration comprise the active ingredient combined with apharmaceutically acceptable carrier, such as sterile water or sterileisotonic saline. Such formulations may be prepared, packaged, or sold ina form suitable for bolus administration or for continuousadministration. Injectable formulations may be prepared, packaged, orsold in unit dosage form, such as in ampules or in multi-dose containerscontaining a preservative. Formulations for parenteral administrationinclude, but are not limited to, suspensions, solutions, emulsions inoily or aqueous vehicles, pastes, and implantable sustained-release orbiodegradable formulations. Such formulations may further comprise oneor more additional ingredients including, but not limited to,suspending, stabilizing, or dispersing agents. In one embodiment of aformulation for parenteral administration, the active ingredient isprovided in dry (i.e., powder or granular) form for reconstitution witha suitable vehicle (e.g., sterile pyrogen-free water) prior toparenteral administration of the reconstituted composition.

[0436] The pharmaceutical compositions may be prepared, packaged, orsold in the form of a sterile injectable aqueous or oily suspension orsolution. This suspension or solution may be formulated according to theknown art, and may comprise, in addition to the active ingredient,additional ingredients such as the dispersing agents, wetting agents, orsuspending agents described herein. Such sterile injectable formulationsmay be prepared using a non-toxic parenterally-acceptable diluent orsolvent, such as water or 1,3-butane diol, for example. Other acceptablediluents and solvents include, but are not limited to, Ringer'ssolution, isotonic sodium chloride solution, and fixed oils such assynthetic mono- or di-glycerides. Other parentally-administrableformulations which are useful include those which comprise the activeingredient in microcrystalline form, in a liposomal preparation, or as acomponent of a biodegradable polymer systems. Compositions for sustainedrelease or implantation may comprise pharmaceutically acceptablepolymeric or hydrophobic materials such as an emulsion, an ion exchangeresin, a sparingly soluble polymer, or a sparingly soluble salt.

[0437] Formulations suitable for topical administration include, but arenot limited to, liquid or semi-liquid preparations such as liniments,lotions, oil-in-water or water-in-oil emulsions such as creams,ointments or pastes, and solutions or suspensions.Topically-administrable formulations may, for example, comprise fromabout 1% to about 10% (w/w) active ingredient, although theconcentration of the active ingredient may be as high as the solubilitylimit of the active ingredient in the solvent. Formulations for topicaladministration may further comprise one or more of the additionalingredients described herein.

[0438] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for pulmonary administrationvia the buccal cavity. Such a formulation may comprise dry particleswhich comprise the active ingredient and which have a diameter in therange from about 0.5 to about 7 nanometers, and preferably from about 1to about 6 nanometers. Such compositions are conveniently in the form ofdry powders for administration using a device comprising a dry powderreservoir to which a stream of propellant may be directed to dispersethe powder or using a self-propelling solvent/powder-dispensingcontainer such as a device comprising the active ingredient dissolved orsuspended in a low-boiling propellant in a sealed container. Preferably,such powders comprise particles wherein at least 98% of the particles byweight have a diameter greater than 0.5 nanometers and at least 95% ofthe particles by number have a diameter less than 7 nanometers. Morepreferably, at least 95% of the particles by weight have a diametergreater than 1 nanometer and at least 90% of the particles by numberhave a diameter less than 6 nanometers. Dry powder compositionspreferably include a solid fine powder diluent such as sugar and areconveniently provided in a unit dose form.

[0439] Low boiling propellants generally include liquid propellantshaving a boiling point of below 65° F. at atmospheric pressure.Generally the propellant may constitute 50 to 99.9% (w/w) of thecomposition, and the active ingredient may constitute 0.1 to 20% (w/w)of the composition. The propellant may further comprise additionalingredients such as a liquid non-ionic or solid anionic surfactant or asolid diluent (preferably having a particle size of the same order asparticles comprising the active ingredient).

[0440] Pharmaceutical compositions of the invention formulated forpulmonary delivery may also provide the active ingredient in the form ofdroplets of a solution or suspension. Such formulations may be prepared,packaged, or sold as aqueous or dilute alcoholic solutions orsuspensions, optionally sterile, comprising the active ingredient, andmay conveniently be administered using any nebulization or atomizationdevice. Such formulations may further comprise one or more additionalingredients including, but not limited to, a flavoring agent such assaccharin sodium, a volatile oil, a buffering agent, a surface activeagent, or a preservative such as methylhydroxybenzoate. The dropletsprovided by this route of administration preferably have an averagediameter in the range from about 0.1 to about 200 nanometers.

[0441] The formulations described herein as being useful for pulmonarydelivery are also useful for intranasal delivery of a pharmaceuticalcomposition of the invention.

[0442] Another formulation suitable for intranasal administration is acoarse powder comprising the active ingredient and having an averageparticle from about 0.2 to 500 micrometers. Such a formulation isadministered in the manner in which snuff is taken, i.e., by rapidinhalation through the nasal passage from a container of the powder heldclose to the nares.

[0443] Formulations suitable for nasal administration may, for example,comprise from about as little as 0.1% (w/w) and as much as 100% (w/w) ofthe active ingredient, and may further comprise one or more of theadditional ingredients described herein.

[0444] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for buccal administration.Such formulations may, for example, be in the form of tablets orlozenges made using conventional methods, and may, for example, 0.1 to20% (w/w) active ingredient, the balance comprising an orallydissolvable or degradable composition and, optionally, one or more ofthe additional ingredients described herein. Alternately, formulationssuitable for buccal administration may comprise a powder or anaerosolized or atomized solution or suspension comprising the activeingredient. Such powdered, aerosolized, or aerosolized formulations,when dispersed, preferably have an average particle or droplet size inthe range from about 0.1 to about 200 nanometers, and may furthercomprise one or more of the additional ingredients described herein.

[0445] A pharmaceutical composition of the invention may be prepared,packaged, or sold in a formulation suitable for ophthalmicadministration. Such formulations may, for example, be in the form ofeye drops including, for example, a 0.1-1.0% (w/w) solution orsuspension of the active ingredient in an aqueous or oily liquidcarrier. Such drops may further comprise buffering agents, salts, or oneor more other of the additional ingredients described herein. Otherophthalmalmically-administrable formulations which are useful includethose which comprise the active ingredient in microcrystalline form orin a liposomal preparation.

[0446] As used herein, “additional ingredients” include, but are notlimited to, one or more of the following: excipients; surface activeagents; dispersing agents; inert diluents; granulating anddisintegrating agents; binding agents; lubricating agents; sweeteningagents; flavoring agents; coloring agents; preservatives;physiologically degradable compositions such as gelatin; aqueousvehicles and solvents; oily vehicles and solvents; suspending agents;dispersing or wetting agents; emulsifying agents, demulcents; buffers;salts; thickening agents; fillers; emulsifying agents; antioxidants;antibiotics; antifungal agents; stabilizing agents; and pharmaceuticallyacceptable polymeric or hydrophobic materials. Other “additionalingredients” which may be included in the pharmaceutical compositions ofthe invention are known in the art and described, for example in Genaro,ed. (1985, Remington's Pharmaceutical Sciences, Mack Publishing Co.,Easton, Pa.), which is incorporated herein by reference.

[0447] Typically, dosages of the compound of the invention which may beadministered to an animal, preferably a human, will vary depending uponany number of factors, including but not limited to, the type of animaland type of disease state being treated, the age of the animal and theroute of administration.

[0448] The compound can be administered to an animal as frequently asseveral times daily,⁰or it may be administered less frequently, such asonce a day, once a week, once every two weeks, once a month, or evenlees frequently, such as once every several months or even once a yearor less. The frequency of the dose will be readily apparent to theskilled artisan and will depend upon any number of factors, such as, butnot limited to, the type and severity of the disease being treated, thetype and age of the animal, etc.

[0449] VIII. Methods

[0450] A. Methods of Identifying Useful Compounds

[0451] The present invention further includes a method of identifying acompound that affects expression of REMODELIN in a cell. The methodcomprises contacting a cell with a test compound and comparing the levelof expression of REMODELIN in the cell so contacted with the level ofexpression of REMODELIN in an otherwise identical cell not contactedwith the compound. If the level of expression of REMODELIN is higher orlower in the cell contacted with the test compound compared to the levelof expression of REMODELIN in the otherwise identical cell not contactedwith the test compound, this is an indication that the test compoundaffects expression of REMODELIN in a cell.

[0452] Similarly, the present invention includes a method of identifyinga compound that reduces expression of REMODELIN in a cell. The methodcomprises contacting a cell with a test compound and comparing the levelof expression of REMODELIN in the cell contacted with the compound withthe level of expression of REMODELIN in an otherwise identical cell,which is not contacted with the compound. If the level of expression ofREMODELIN is lower in the cell contacted with the compound compared tothe level in the cell that was not contacted with the compound, thenthat is an indication that the test compound affects reduces expressionof REMODELIN in a cell.

[0453] One skilled in the art would appreciate, based on the disclosureprovided herein, that the level of expression of REMODELIN in the cellcan be measured by determining the level of expression of mRNA encodingREMODELIN. Alternatively, the level of expression of mRNA encodingREMODELIN can be determined by using immunological methods to assessREMODELIN production from such mRNA as exemplified herein using Westernblot analysis using an anti-REMODELIN antibody of the invention.Further, nucleic acid-based detection methods, such as Northern blot andPCR assays and the like, can be used as well. In addition, the level ofREMODELIN activity in a cell can also be assessed by determining thelevel of various parameters which can be affected by REMODELIN activitysuch as, for example, the level of cell proliferation and/or migration,the level of expression in adventitia, the level of adventitialfibrosis, the level of fibrosis in other organs (e.g., lung, liver,among others), the level of arterial restenosis, the level ofossification, the level of bone formation and fracture healing, thelevel of osteoblast proliferation, and the like. Thus, one skilled inthe art would appreciate, based upon the extensive disclosure andreduction to practice provided herein, that there are a plethora ofmethods which can be used to asses the level of expression of REMODELINin a cell including those methods disclosed herein, methods well-knownin the art, and other methods to be developed in the future.

[0454] Further, one skilled in the art would appreciate based on thedisclosure provided herein that, as disclosed in the examples below, acell which lacks endogenous REMODELIN expression can be transfected witha vector comprising an isolated nucleic acid encoding REMODELIN wherebyexpression of REMODELIN is effected in the cell. The transfected cell isthen contacted with the test compound thereby allowing the determinationof whether the compound affects the expression of REMODELIN. Therefore,one skilled in the art armed with the present invention would be ableto, by selectively transfecting a cell lacking detectable levels ofREMODELIN using REMODELIN-expressing vectors, identify a compound whichselectively affects REMODELIN expression.

[0455] The skilled artisan would further appreciate, based upon thedisclosure provided herein, that where an isolated nucleic acid encodingREMODELIN is administered to a cell lacking endogenous detectable levelsof REMODELIN expression such that detectable REMODELIN is produced bythe cell, the isolated nucleic acid can comprise an additional nucleicacid encoding, e.g., a tag polypeptide, covalently linked thereto. Thisallows the detection of REMODELIN expression by detecting the expressionof the tag polypeptide. Thus, the present invention encompasses methodsof detecting REMODELIN expression by detecting expression of anothermolecule which is co-expressed with REMODELIN.

[0456] The invention includes a method of identifying a protein thatspecifically binds with REMODELIN. That is, one skilled in the art wouldappreciate, based upon the disclosure provided herein, that REMODELIN,which comprises several myristoylation domains. Further, REMODELINcomprises a putative signal peptide indicating the molecule can besecreted from a cell. These data indicate that REMODELIN likely effectsits biological function(s) by specifically binding with at least oneprotein, preferably a REMODELIN receptor, another REMODELIN molecule,and/or a REMODELIN ligand. Thus, the invention encompasses methods,which are well-known in the art or to be developed, for identifying aprotein that specifically binds with and/or associates with REMODELIN.Such methods include, but are not limited to, protein binding assayswherein the target protein, i.e., REMODELIN, is immobilized on anappropriate support and incubated under conditions that allow REMODELINbinding with a REMODELIN-associated protein. REMODELIN can beimmobilized on a support using standard methods such as, but not limitedto, production of REMODELIN comprising a glutathione-S-transferase (GST)tag, a maltose binding protein (MBP) tag, or a His₆-tag, where thefusion protein is then bound to glutathione-Sepharose beads, amaltose-column, or a nickel chelation resin (e.g., His-Bind resin,Novagen, Madison, Wis.), respectively. The solid support is washed toremove proteins which may be non-specifically bound thereto and anyREMODELIN-associated protein can then be dissociated from the matrixthereby identifying a REMODELIN-associated protein.

[0457] In addition, a protein that specifically binds with REMODELIN,e.g., a receptor, a ligand, and/or other REMODELIN-associated protein,can be identified using, for example, a yeast two hybrid assay. Yeasttwo hybrid assay methods are well-known in the art and can be performedusing commercially available kits (e.g., MATCHMAKER™ Systems, ClontechLaboratories, Inc., Palo Alto, Calif., and other such kits) according tostandard methods. Therefore, once armed with the teachings providedherein, e.g., the full amino and nucleic acid sequences of the “bait”protein, REMODELIN, one skilled in the art can easily identify a proteinthat specifically binds with REMODELIN such as, but not limited to, aREMODELIN receptor protein, a REMODELIN ligand, and the like.

[0458] One skilled in the art would understand, based upon thedisclosure provided herein, that the invention encompasses any moleculeidentified using the methods discussed elsewhere herein. That is,molecules that associate with REMODELIN, such as but not limited to, aREMODELIN receptor protein, a REMODELIN ligand protein, or both, can beused to develop therapeutics and diagnostics for diseases, disorders orconditions mediated by REMODELIN interaction with a REMODELIN-associatedprotein such as negative remodeling, arterial restenosis, adventitialfibrosis, excessive wound healing responses, scarring, keloids,excessive bone formation, fracture healing, ectopic ossification,excessive fibrous tissue formation, failure of dorsal closure, spinabifida-like effects, and the like. That is, one skilled in the art wouldappreciate, as more fully set forth elsewhere herein in discussingantibodies that specifically bind with REMODELIN, that aREMODELIN-associated protein can be used to develop therapeutics thatinhibit REMODELIN activity in a cell by inhibiting necessary REMODELINreceptor/ligand interactions and other REMODELIN binding interactions,which are required for REMODELIN activity.

[0459] REMODELIN-associated proteins identified by the above-disclosedmethods can be used directly to inhibit REMODELIN interactions bycontacting a cell with the REMODELIN-associated protein, or a portionthereof, or they can be used to develop antibodies and/orpeptidomimetics that can inhibit the REMODELIN-associated interactionwith REMODELIN thereby inhibiting REMODELIN function and activity. Thus,REMODELIN-associated proteins, including a REMODELIN receptor/ligandprotein, are useful and are encompassed by the invention.

[0460] B. Methods of Treating or Alleviating a Disease, Disorder orCondition Associated with or Mediated by REMODELIN Expression

[0461] The invention includes a method of alleviating a disease,disorder or condition mediated by altered expression of REMODELIN. Themethod comprises administering an antisense nucleic acid complementaryto a nucleic acid encoding REMODELIN to a patient afflicted with adisease, disorder or condition mediated by increased REMODELINexpression compared to the level of REMODELIN expression in otherwiseidentical but normal tissue, i.e., tissue which does not exhibit anydetectable clinical parameters associated with the disease, disorder orcondition being treated or alleviated. This, in turn, mediates adecrease in REMODELIN expression thereby alleviating a disease, disorderor condition mediated by abnormal expression of REMODELIN. Suchdiseases, disorder or conditions include, but are not limited to,negative remodeling, arterial restenosis, adventitial fibrosis,excessive wound healing responses, scarring, keloids, abnormal boneformation and/or bone density, fracture healing, ectopic ossification,excessive fibrous tissue formation, lack of dorsal closure, spinabifida-like conditions, osteogenesis imperfecta (OI), dystrophicepidermolysis bullosea (DEB), Bethlem myopathy, tissue calcification,including, but not limited to, calcification of implants, such as heartvalves, and the like.

[0462] The data disclosed herein demonstrate that REMODELIN expressionis induced in fibroblasts following an injury. As such, REMODELIN ispart of any wound healing process which is characterized by granulationtissue formation, proliferation, and migration of fibroblasts withsubsequent apoptosis of these cells extracellular matrix production.Although the wound healing response/process is a normal physiologicresponse to injury, there are many conditions where an excessive woundhealing response leads to symptoms or disease. Thus, the skilled artisanwould appreciate, based upon the disclosure provided herein, thatbecause the wound healing response is similar for all organs andtissues, the effects of expression of REMODELIN are similar independentof where the injury occurred. Excessive wound healing would result invarious conditions depending on the situs of injury such that, forexample, in an artery, excessive wound healing would result in negativeremodeling with loss of vessel diameter due to adventitial fibrosis. Theformation of scars and keloids is an excessive fibrotic reactionassociated with excessive wound healing. Chronic inflammatory conditionsoften lead to organ fibrosis, e.g., liver fibrosis and lung fibrosis.

[0463] The data disclosed herein demonstrate that when REMODELIN isinhibited using, for example, but not limited to, an antisense nucleicacid, the cell exhibited an altered morphology indicating decreased celladhesion to the substratum and decreased cell-cell interaction. Further,the data disclosed herein demonstrate that REMODELIN antisenseexpression is associated with and/or mediates cell turnover. Therefore,the data further indicate that REMODELIN is involved in and/or isassociated with processes involving cell turnover, cell adhesion andcell-cell interaction such as, but not limited to, negative remodeling,adventitial fibrosis, and the like.

[0464] An example of a disease, disorder or a condition associated withor mediated by REMODELIN expression is, organ and tissue fibrosis (e.g.,adventitial, lung, liver, and retroperitoneal fibrosis, and the like),hypertrophic scars, keloids, excessive bone formation, ectopicossification, and the like.

[0465] Although inhibition of REMODELIN is exemplified by administeringan antisense to a cell thereby inhibiting expression of REMODELIN in thecell, one skilled in the art would appreciate that there are a wideplethora of methods for inhibiting protein expression and/or activity ina cell. Such methods include, but are not limited to, inhibitingexpression of REMODELIN using ribozymes, and inhibiting activity of theprotein in a cell by, for instance, administering an antibody to thecell by, e.g., administering a nucleic acid encoding the antibody to thecell such that the antibody is expressed in the cell thus delivering theantibody to the cell cytosol. The use of these “intrabodies” to inhibitthe intracellular activity of a protein are well-known in the art. See,e.g., Verma et al. (1997, Nature 389:239-242; Benhar & Pastan, 1995, J.Biol. Chem. 270:23373-23380; Willuda et al., 1999, Cancer Res.59:5758-5767; and Worn et al., 2000, J. Biol. Chem. 275:2795-2803).Therefore, the present invention encompasses any method of inhibitingthe activity of a protein of interest in a cell using such methods asare known in the art or to be developed in the future.

[0466] The invention includes a method of treating bone disease in amammal by inhibiting or decreasing expression of REMODELIN. This isinhibiting e the data disclosed herein demonstrate that inhibitingREMODELIN in osteoblasts mediates a powerful effect thereby suggesting arole for this gene in bone formation and indicating a role for REMODELINin certain bone disease. That is, bone disease can be mediated by and/orassociated with bone formation at undesirable sites, such as aftertrauma, leading to ossification of otherwise non-calcifying tissues, forexample, skeletal muscle and vascular tissues. Indeed, calcification isan important problem involving prostheses and implants, such as, but notlimited to, heart valves. Therefore, the data disclosed herein suggestthat REMODELIN expression plays a role in bone formation, ossification,and calcification in response to trauma and/or injury such that methodsaffecting that expression can treat a disease, such as a bone disease,mediated by or associated with those REMODELIN-associated processes.

[0467] The data presented herein further indicates that over-expressionof REMODELIN mediates and/or is associated with lack of dorsal closure,abnormal bone density and bone growth, including dwarfism, and spinabifida-like phenotype. Thus, these data demonstrate that a method ofinhibiting or decreasing REMODELIN expression can be useful for treatingdiseases associated with or mediated by over-expression of REMODELINsuch as, but not limited to, treatment of lack of dorsal closure,abnormal bone density, dwarfism, spina bifida-like phenotype, and thelike. Thus, the data disclosed herein suggest that REMODELIN plays animportant role(s) including, but not limited to, a role in boneformation, bone density, dorsal closure, inhibition of calcification,and the like, and that any disease, disorder or condition associatedwith those processes can be treated or alleviated by any treatmentdecreasing or inhibiting the level of REMODELIN expression compared withthe level of REMODELIN in the absence of such treatment.

[0468] One skilled in the art would appreciate, based upon thedisclosure provided herein, that conditions associated with prematurecalcification or mineralization of bone and other tissues, includingvascular calcification, and calcification of transplants, including, butnot limited to, heart valves, can be treated or ameliorated byinhibiting REMODELIN expression. This is because the data disclosedherein demonstrate that REMODELIN expression is associated with, ormediates, bone mineralization and calcification such that inhibitingsuch expression can inhibit those processes. Inhibition of calcificationprovides an important therapeutic benefit for treatment of conditionsassociated with, or mediated by, premature ossification or unwantedcalcification generally, such conditions being well-known in the art.

[0469] The invention includes a method of treating cartilage disease ina mammal. This is because, as demonstrated by the data disclosedelsewhere herein, lack of REMODELIN expression can lead to prematurecalcification of the cartilage matrix thereby inhibiting bone growth.Thus, the present invention encompasses a method of treating cartilagedisease wherein the level of REMODELIN in a cell, tissue or organ in apatient suffering from cartilage disease, is increased relative to thelevel of REMODELIN in a cell from the patient prior to, or in theabsence of, treatment or detectably increased with respect to the levelof REMODELIN in an otherwise identical cell obtained from a mammal notsuffering from any disease, disorder or condition. Therefore, thepresent invention includes methods to treat cartilage disease byincreasing REMODELIN expression.

[0470] One skilled in the art would understand, based upon thedisclosure provided herein, that reducing expression of REMODELIN canmediate a beneficial effect in a patient afflicted with excessive woundhealing resulting in negative remodeling, adventitial fibrosis, fibrosisin any organ or tissue (e.g., liver fibrosis and lung fibrosis),scarring, keloids, fibrotic reaction associated with excessive woundhealing, excessive bone formation, ectopic ossification, and the like.Thus, decreased REMODELIN expression can be useful for treating suchdiseases, disorders, or conditions, and the present inventionencompasses such methods of treating these diseases by inhibiting thelevel of REMODELIN in a cell. This is because, as disclosed elsewhereherein, increased expression of REMODELIN is associated with abnormalcell proliferation associated with arterial restenosis, negativeremodeling, adventitial fibrosis, fibrosis in any organ or tissue (e.g.,liver, lung, among others), excessive bone formation, ectopicossification, altered cell adhesion and cell-cell interaction, cellturnover, and the like. Thus, one skilled in the art would appreciate,based upon the disclosure provided herein, that inhibition of REMODELINexpression can inhibit the deleterious effects of REMODELIN abnormalexpression.

[0471] One skilled in the art would understand, based upon thedisclosure provided herein, that since reduced REMODELIN expression canmediate a beneficial effect, methods of decreasing expression ofREMODELIN, decreasing the stability of the protein in the cytoplasm,decreasing the level of REMODELIN polypeptide present in the cell,and/or decreasing the activity of REMODELIN in a cell (using, e.g.,antisense nucleic acids, ribozymes, antibodies, and the like), can beused to treat and/or alleviate a disease, disorder or conditionassociated with altered expression of REMODELIN where a lower level ofREMODELIN relative to the level of REMODELIN in that cell prior to, orin the absence of, treatment provides a benefit. A benefit such aspreventing decreased bone growth and/or density, failure of dorsalclosure, and spina bifida-like phenotype such as are associated withover-expression of REMODELIN as disclosed elsewhere herein. Thus,whether an antisense nucleic acid or a blocking antibody isadministered, the present invention includes a method where theexpression of REMODELIN be reduced in a cell compared with an otherwisecell, to treat/or alleviate preventing decreased bone growth and/ordensity, failure of dorsal closure, and spina bifida-like phenotype.

[0472] Techniques for inhibiting expression of a nucleic acid in a cellare well known in the art and encompass such methods as disclosed herein(e.g., inhibition using an antibody, an antisense nucleic acid, and thelike). Other techniques useful for inhibiting expression of a nucleicacid encoding REMODELIN include, but are not limited to, usingnucleotide reagents that target specific sequences of the REMODELINpromoter, and the like.

[0473] Antisense nucleic acids that inhibit expression of REMODELIN,among other things, can be used for the manufacture of a medicament fortreatment of a disease, disorder or condition mediated by increasedexpression of REMODELIN, when compared with expression of REMODELIN in acell and/or a patient not afflicted with the disease, disorder orcondition.

[0474] One skilled in the art would understand, based upon thedisclosure provided herein, that it may be useful to increase the levelor activity of REMODELIN in a cell. That is, the data disclosed hereindemonstrating the association between REMODELIN expression and woundhealing and bone growth, indicate that overexpression or an increase inREMODELIN activity can accelerate wound healing and bone growth. Thiscan be useful to treat or alleviate a disease, disorder of conditionassociated with or mediated by decreased expression, level, or activityof REMODELIN (e.g., various forms of organ and tissue damage includingbone fractures), when compared to the expression, level or activity ofREMODELIN in otherwise identical cell, tissue, or animal that does notsuffer from the disease, disorder or condition, by administeringREMODELIN. Such diseases, disorders or conditions include, but are notlimited to, tissue damage or injury, including bone fracture, woundhealing, and the like, and collagen-related diseases.

[0475] The data disclosed herein suggest that over- and under-expressionof REMODELIN are associated with disease, disorder or condition suchthat a method of decreasing or increasing the level of REMODELIN canproduce a benefit. Therefore, a method of affecting the level ofREMODELIN to treat/alleviate a wide plethora of diseases, disorders, orconditions is disclosed herein, where the level of REMODELIN is eitherdecreased or increased compared with the level of REMODELIN in the cellprior to treatment, or compared with the level of REMODELIN in anotherwise identical cell that is obtained from a mammal known not to beafflicted with a disease, disorder or condition associated with, ormediated by, an altered level of REMODELIN.

[0476] The skilled artisan would understand, based upon the disclosureprovided herein, that both increasing and decreasing REMODELINexpression and/or activity in a cell, tissue or organ can treat adisease, disorder or condition associated with REMODEL expression oractivity. This is because the data disclosed herein demonstrate that incertain instances, decreasing REMODELIN expression provides a benefitwhile, in other instances, increasing REMODELIN expression istherapeutic, e.g., by accelerating wound healing, bone formation, andthe like. Therefore, the present invention encompasses methods oftreating and/or alleviating conditions associated with either increasedor decreased REMODELIN expression, and the skilled artisan, armed withthe disclosure provided herein, would appreciate which conditions wouldbenefit from decreasing or increasing REMODELIN expression and/oractivity in a cell in a mammal.

[0477] Whether expression of REMODELIN, levels of the polypeptide, orits activity, is increased or decreased, one skilled in the art wouldappreciate, based on this disclosure, that methods of reducing orinducing REMODELIN of the invention encompass administering arecombinant cell that either expresses or lacks expression of REMODELIN.Thus, one skilled in the art would appreciate, based on the disclosureprovided herein, that the present invention encompasses cell and genetherapy methods to effect either a detectable increase or decrease inthe level of REMODELIN expression in a mammal.

[0478] In another embodiment of the invention, an individual sufferingfrom a disease, disorder or a condition that is associated with ormediated by REMODELIN expression can be treated by supplementing,augmenting and/or replacing defective cells with cells that lackREMODELIN expression. The cells can be derived from cells obtained froma normal syngeneic matched donor or cells obtained from the individualto be treated. The cells may be genetically modified to inhibitREMODELIN expression. Alternatively, the cells can be modified toincrease REMODELIN expression using recombinant methods well-known inthe art. Also, the invention encompasses using normal cells obtainedfrom an otherwise identical donor that does not suffer from any diseaseor disorder associated with altered REMODELIN expression, which cellscan be administered to a mammal in need thereof.

[0479] Additionally, the invention includes ex vivo techniques where acell is obtained from the mammal, modified to express increased ordecreased level of REMODELIN, and reintroduced into the mammal.Moreover, cells from the mammal which express a normal level ofREMODELIN, compared with the level of REMODELIN expressed in anotherwise identical cell obtained from a like mammal not suffering fromany condition associated with altered REMODELIN expression, can be grownand expanded and an effective number of the cells can be reintroducedinto the mammal. Such methods include cell and gene therapy techniquesrelating to use of bone marrow stromal cells which methods arewell-known in the art. Thus, one skilled in the art would appreciatethat cell therapy and gene therapy relating to cells that have or lackdetectable REMODELIN expression wherein the cells are administered invivo are encompassed in the present invention.

[0480] The data presented herein demonstrate that REMODELIN expressioncan be modulated using TGF-β and TGF-β receptor. Specifically, TGF-βinduces expression of REMODELIN, and TGF-β receptor reduces expressionof REMODELIN perhaps by, and without wishing to be bound by anyparticular theory, competing for the free REMODELIN thereby inhibitingthe ligand/receptor interaction between TGF-β and its receptorassociated with an increase in the level of REMODELIN expression. Thus,one skilled in the art would understand, based upon the disclosureprovided herein, that TGF-β or TGF-β receptor can be administered to amammal to treat a disease, disorder or a condition associated withaltered expression of REMODELIN in a mammal.

[0481] Accordingly, one skilled in the art, armed with the disclosureprovided herein, would appreciate that the invention includes a methodof increasing REMODELIN expression in a mammal by administering TGF-β.This is because, as discussed previously elsewhere herein, and as wouldbe appreciated by the routineer based upon the disclosure providedherein, interaction of TGF-β with its receptor, i.e., TGF-β receptortype II, increases the level of REMODELIN expression in a cell contactedwith TGF-β. Therefore, where increased level of REMODELIN expressionprovides a benefit, as more fully discussed elsewhere herein,administering TGF-β, which increases the level of REMODELIN expressioncompared with the level of REMODELIN expression prior to, or in theabsence of, TGF-β, is desired and provides a therapeutic benefit.

[0482] Alternatively, one skilled in the art would understand, basedupon the disclosure provided herein, that the invention includes amethod of reducing REMODELIN expression in a mammal by administeringTGF-β receptor type II. This is because, as noted previously elsewhereherein and without wishing to be bound by any particular theory, thedata disclosed herein demonstrate that interaction between TGB-β and itsreceptor, TGF-β receptor II, mediates an increase in the level ofREMODELIN expression in a cell such that inhibiting that interaction byadministering free, soluble TGF-β receptor type II can reduce the levelof interaction of TGF-β with a TGF-β receptor type II on the surface ofa cell, thereby reducing and/or preventing the increase in the level ofREMODELIN expression mediated by TGF-β that would otherwise occur in theabsence of the excess receptor. Thus, administration of TGF-β receptortype II decreases the level of REMODELIN expression, and can be used totreat any disease, disorder or condition associated with, or mediatedby, increased level of REMODELIN such as, but not limited to, . . .

[0483] The skilled artisan would further appreciate, based upon thedisclosure provided herein, that a TGF-β receptor type II antagonist isincluded in the invention. That is, any compound that specificallyinhibits transmission of a signal via the TGF-β receptor type II can beused to inhibit expression of REMODELIN. Such TGF-β antagonists include,but are not limited to, any molecule that inhibits TGF-β interactionwith the receptor such as an antibody that sterically inhibitsligand/receptor interaction between TGF-β and TGF-β receptor type II.Such molecules, by inhibiting necessary interaction between TGF-β andthe TGF-β receptor type II, inhibit TGF-β-associated increase inREMODELIN expression thereby inhibiting the effect(s) of increasedREMODELIN expression in cell processes. TGF-β receptor type IIantagonists are well-known in the art and the invention furtherencompasses such antagonists as are discovered in the future.

[0484] The skilled artisan would also understand, based upon thedisclosure provided herein, that the amount of inhibitor administered toa mammal in order to inhibit REMODELIN expression can be easily assessedby determining the level of REMODELIN expression before and afteradministration of the inhibitor. Preferably, inhibition of REMODELINexpression mediated by TGF-β using soluble TGF-β receptor type IIcomprises administering about 2 mg/kg of soluble receptor; however, thepresent invention is not limited to this dose, and the skilled artisanwould be able to readily determine, based upon the disclosure providedherein, the appropriate dose for each mammal depending on variousparameters, including, but not limited to, the level of REMODELINexpression detected in the mammal prior to and during treatment.

[0485] The present invention encompasses increasing REMODELIN expressionby administering TGF-β to a mammal. This is because, as set forthpreviously elsewhere herein, contacting a cell with TGF-β increasesREMODELIN expression and increased REMODELIN expression can providetherapeutic benefit to the mammal, including, but not limited to,increased bone formation, accelerated wound healing, and the like. Thus,the skilled artisan would understand, based upon the disclosure providedherein, that the present invention includes increasing the level ofREMODELIN expression by administering TGF-β to a mammal.

[0486] The invention includes a method of affecting cellular geneexpression by administering an isolated nucleic acid encoding REMODELINor an isolated nucleic acid antisense to a nucleic acid encodingREMODELIN. This is because, as the data presented herein demonstrate,expression of a nucleic acid encoding REMODELIN or expression of anucleic acid antisense to a nucleic acid encoding REMODELIN affects thelevel of several cellular genes including, but not limited to, TGF-β1,collagen IIIα1, ostiopontin, biglycan, alkaline phosphatase (ALP), andbone morphogenic protein 4 (BMP-4). Thus, increasing the level ofREMODELIN in a cell, such as by, among other things, administering anucleic acid encoding REMODELIN to the cell and expressing the REMODELINtherefrom under the control of a promoter that drives increased level ofexpression, can be used to control the expression of various proteins,including, but not limited to, TGF-β1, collagen IIIα1, ostiopontin,biglycan, ALP and BMP-4, where affecting the expression of theseproteins provides a benefit to a mammal.

[0487] The data disclosed herein demonstrate that REMODELIN can inhibitexpression of bone differentiation markers thereby inhibitingcalcification/ossification. Thus, the skilled artisan would appreciate,based upon the disclosure provided herein, that REMODELIN, by inhibitingbone differentiation markers, can inhibit prematurecalcification/ossification. Therefore, the present invention encompassesa method of inhibiting calcification comprising increasing expression ofREMODELIN in a cell. This is because the data disclosed hereindemonstrate that expression of REMODELIN inhibits bone differentiationmarkers thereby inhibiting calcification.

[0488] In addition to replacing defective cells with repaired cells ornormal cells from syngeneic, immunologically-matched donors, the methodof the invention may also be used to facilitate expression of a desiredprotein that when secreted in the an animal, has a beneficial effect.That is, cells may be isolated, furnished with a gene encoding REMODELINand introduced into the donor or into a syngeneic matched recipientwherein expression of exogenous REMODELIN exerts a therapeutic effect.

[0489] One skilled in the art would understand, based upon thedisclosure provided herein, that secretion of REMODELIN from a cell iscontemplated in the present invention. That is, the routineer wouldappreciated, based upon the disclosure provided herein, that secretionof REMODELIN from a cell can be a useful therapeutic method and that thepresent invention includes secretion of REMODELIN from a cell. Secretionof REMODELIN from a cell can be effected according to standard methodswell-known in the art and methods to be developed in the future. Suchmethods include, but are not limited to, covalently linking a nucleicacid encoding a signal peptide of a secreted molecule (e.g., insulin;MALLVHFLPLLALLALWEPKPTQA [SEQ ID NO: 8]) to the 5′ end of an isolatednucleic acid encoding REMODELIN. A wide plethora of signal sequencesthat can be used to mediate secretion of a protein from a cell areavailable and well-known in the art and the invention includes those aswell as sequences to be developed in the future to drive secretion of aprotein from a cell.

[0490] This aspect of the invention relates to gene therapy in whichtherapeutic amounts of REMODELIN are administered to an individual. Thatis, according to some aspects of the present invention, recombinantcells transfected with either nucleic acid encoding REMODELIN, antisensenucleic acids, or a knock-out targeting vector of the invention, can beused as cell therapeutics to treat a disease, disorder or a conditioncharacterized by altered expression of REMODELIN, including the lack ofexpression of REMODELIN.

[0491] In particular, a gene construct that comprises a heterologousgene which encodes REMODELIN is introduced into cells. These recombinantcells are used to purify isolated REMODELIN, which was is administeredto an animal. One skilled in the art would understand, based upon thedisclosure provided herein, that instead of administering an isolatedREMODELIN polypeptide, REMODELIN can be administered to a mammal in needthereof by administering to the mammal the recombinant cells themselves.This will benefit the recipient individual who will benefit when theprotein is expressed and secreted by the recombinant cell into therecipient's system.

[0492] According to the present invention, gene constructs comprisingnucleotide sequences of the invention are introduced into cells. Thatis, the cells, referred to herein as “recombinant cells,” aregenetically altered to introduce a nucleic acid encoding REMODELIN or anucleic acid that inhibits REMODELIN expression in and/or secretion bythe recombinant cell (e.g., an antisense REMODELIN nucleic acid, anucleic acid encoding an anti-REMODELIN antibody, TGF-β receptor, andthe like), thereby mediating a beneficial effect on an recipient towhich the recombinant cell is administered. According to some aspects ofthe invention, cells obtained from the same individual to be treated orfrom another individual, or from a non-human animal, can be geneticallyaltered to replace a defective REMODELIN gene and/or to introduce aREMODELIN gene whose expression has a beneficial effect on theindividual, or to inhibit REMODELIN expression which can have abeneficial effect on the individual.

[0493] In some aspects of the invention, an individual suffering from adisease, disorder or a condition can be treated by supplementing,augmenting and/or replacing defective or deficient nucleic acid encodingREMODELIN by providing an isolated recombinant cells containing geneconstructs that include normal, functioning copies of a nucleic acidencoding REMODELIN. This aspect of the invention relates to gene therapyin which the individual is provided with a nucleic encoding REMODELINfor which they are deficient in presence and/or function. The isolatednucleic acid encoding REMODELIN provided by the cell compensates for thedefective REMODELIN expression of the individual, because, when thenucleic acid is expressed in the individual, a protein is produced whichserves to alleviate or otherwise treat the disease, disorder orcondition in the individual. Such nucleic acid preferably encodes aREMODELIN polypeptide that is secreted from the recombinant cell.

[0494] In all cases in which a gene construct encoding REMODELIN istransfected into a cell, the nucleic acid is operably linked to anappropriate promoter/regulatory sequence which is required to achieveexpression of the nucleic acid in the recombinant cell. Suchpromoter/regulatory sequences include but are not limited to,constitutive and inducible and/or tissue specific and differentiationspecific promoters, and are discussed elsewhere herein. Constitutivepromoters include, but are not limited to, the cytomegalovirus immediateearly promoter and the Rous sarcoma virus promoter. In addition,housekeeping promoters such as those which regulate expression ofhousekeeping genes may also be used. Other promoters include those whichare preferentially expressed in cells of the central nervous system,such as, but not limited the promoter for the gene encoding glialfibrillary acidic protein. In addition, promoter/regulatory elements maybe selected such that gene expression is inducible. For example, atetracycline inducible promoter may be used (Freundlich et al., 1997,Meth. Enzymol. 283:159-173).

[0495] The gene construct is preferably provided as an expression vectorwhich includes the coding sequence of a mammalian REMODELIN of theinvention operably linked to essential promoter/regulatory sequencessuch that when the vector is transfected into the cell, the codingsequence is expressed by the cell. The coding sequence is operablylinked to the promoter/regulatory elements necessary for expression ofthe sequence in the cells. The nucleotide sequence that encodes theprotein may be cDNA, genomic DNA, synthesized DNA or a hybrid thereof oran RNA molecule such as mRNA.

[0496] The gene construct, which includes the nucleotide sequenceencoding REMODELIN operably linked to the promoter/regulatory elements,may remain present in the cell as a functioning episomal molecule or itmay integrate into the chromosomal DNA of the cell. Genetic material maybe introduced into cells where it remains as separate genetic materialin the form of a plasmid. Alternatively, linear DNA which can integrateinto a host cell chromosome may be introduced into the cell. Whenintroducing DNA into the cell, reagents which promote DNA integrationinto chromosomes may be added. DNA sequences which are useful to promoteintegration may also be included in the DNA molecule. Alternatively, RNAmay be introduced into the cell.

[0497] In order for genetic material in an expression vector to beexpressed, the promoter/regulatory elements must be operably linked tothe nucleotide sequence that encodes the protein. In order to maximizeprotein production, promoter/regulatory sequences may be selected whichare well suited for gene expression in the desired cells. Moreover,codons may be selected which are most efficiently transcribed in thecell. One having ordinary skill in the art can produce recombinantgenetic material as expression vectors which are functional in thedesired cells.

[0498] It is also contemplated that promoter/regulatory elements may beselected to facilitate tissue specific expression of the protein. Thus,for example, specific promoter/regulatory sequences may be provided suchthat the heterologous gene will only be expressed in the tissue wherethe recombinant cells are implanted. Additionally, the skilled artisanwould appreciate, based upon the disclosure provided herein, that theREMODELIN promoter can be operably linked to a nucleic acid of interestthereby directing the expression of the nucleic acid at the site oftissue or organ injury and wounding. More specifically, the REMODELINpromoter can be used, but is not limited, to direct expression of anangiogenic growth factor to promote angiogenesis after myocardialinfarction. Similarly, the REMODELIN promoter can drive expression of anucleic acid of interest where such expression is beneficial wheretissue ischemia and impaired wound healing are a problem (e.g.,ulcerations of the skin, and the like).

[0499] One skilled in the art would understand, based upon thedisclosure provided herein, that the preferred tissues where theexpression or lack of expression of REMODELIN is to be targeted include,but are not limited to, ulcerations of the skin, bone fractures, and thelike. In addition, promoter/regulatory elements may be selected suchthat gene expression is inducible. For example, a tetracycline induciblepromoter may be used (Freundlich et al., 1997, Meth. Enzymol.283:159-173).

[0500] Without wishing to be bound by any particular theory, the nucleicacid encoding REMODELIN preferably includes a putative signal sequenceas disclosed elsewhere herein (e.g., amino acids 1 to 32 of humanREMODELIN; SEQ ID NO: 3) and amino acids 1 to 32 of rat REMODELIN_(S)(SEQ ID NO: 1), which may direct the transport and secretion of theREMODELIN encoded by the isolated nucleic acid in the recombinant cell.The signal sequence is likely processed and removed upon secretion ofthe mature REMODELIN protein from the cell. Alternatively, withoutwishing to be bound by any particular theory, the putative signalsequence may not be cleaved, but may instead be a transmembrane domain.

[0501] In addition to providing cells with recombinant genetic materialthat either corrects a genetic defect in the cells, that encodes aprotein which is otherwise not present in sufficient quantities and/orfunctional condition so that the genetic material corrects a geneticdefect in the individual, and/or that encodes a protein which is usefulas beneficial in the treatment or prevention of a particular disease,disorder or condition associated therewith, and that inhibits expressionof REMODELIN in the cell (e.g., a knock-out targeting vector, anantisense nucleic acid, and the like), genetic material can also beintroduced into the recombinant cells used in the present invention toprovide a means for selectively terminating such cells should suchtermination become desirable. Such means for targeting recombinant cellsfor destruction may be introduced into recombinant cells.

[0502] According to the invention, recombinant cells can be furnishedwith genetic material which renders them specifically susceptible todestruction. For example, recombinant cells may be provided with a genethat encodes a receptor that can be specifically targeted with acytotoxic agent. An expressible form of a gene that can be used toinduce selective cell death can be introduced into the recombinantcells. In such a system, cells expressing the protein encoded by thegene are susceptible to targeted killing under specific conditions orin, the presence or absence of specific agents. For example, anexpressible form of a herpes virus thymidine kinase (herpes tk) gene canbe introduced into the recombinant cells and used to induce selectivecell death. When the introduced genetic material that includes theherpes tk gene is introduced into the individual, herpes tk will beproduced. If it is desirable or necessary to kill the implantedrecombinant cells, the drug gangcyclovir can be administered to theindividual which will cause the selective killing of any cell producingherpes tk. Thus, a system can be provided which allows for the selectivedestruction of implanted recombinant cells.

[0503] One skilled in the art would understand, based upon thedisclosure provided herein, that the present invention encompassesproduction of recombinant cells to either provide REMODELIN to orinhibit REMODELIN expression in a mammal. That is, the cells can be usedto administer REMODELIN to an animal or to deliver a molecule (e.g., aknock-out targeting vector, an antisense nucleic acid, a ribozyme, andantibody that specifically binds with REMODELIN, and the like).

[0504] Administration of REMODELIN to an animal can be used as a modelsystem to study the mechanism of action of REMODELIN or to develop modelsystems useful for the development of diagnostics and/or therapeuticsfor diseases, disorders or conditions associated with REMODELINexpression.

[0505] Further, the delivery of REMODELIN to an animal mediated byadministration of recombinant cells expressing and secreting REMODELINcan also be used to treat or alleviate a disease, disorder or conditionwhere increasing the level of REMODELIN mediates a therapeutic effect.More specifically, administration of REMODELIN to an animal byadministering a recombinant cell expressing a nucleic acid encodingREMODELIN can be useful for treatment of impaired wound healing, bonefracture, and impaired bone formation, among other things.

[0506] Alternatively, administration of recombinant cells comprising anucleic acid the expression of which inhibits or reduces REMODELINexpression, activity, and/or secretion from a cell, can be used as amodel for the development of diagnostics and/or therapeutics useful fordiseases, disorders or conditions associated with or mediated byREMODELIN expression, activity, and/or secretion. The present inventionencompasses that the recombinant cells can produce the molecule thatinhibits REMODELIN expression thereby providing such molecule to theanimal. Alternatively, without wishing to be bound by any particulartheory, the recombinant cells themselves, which are otherwise functionalcells, except for the inability to express REMODELIN, can perform thefunctions of otherwise identical but non-recombinant cells, withoutbeing subject to the REMODELIN signaling pathway.

[0507] Cells, both obtained from an animal, from established cell linesthat are commercially available or to be developed, or primary cellscultured in vitro, can be transfected using well known techniquesreadily available to those having ordinary skill in the art. Thus, thepresent invention is not limited to obtaining cells from a donor animalor from the patient animal itself. Rather, the invention includes usingany cell that can be engineered using a nucleic acid of the inventionsuch that the recombinant cell either expresses REMODELIN (where it didnot express REMODELIN prior to being engineered, or where the cellproduced REMODELIN at a different level prior to the introduction of thenucleic acid into the cell) or the recombinant cell does not expressREMODELIN or expresses it at a lower level (where it expressed REMODELINbefore or expressed REMODELIN at a different level prior to introductionof the nucleic acid into the cell).

[0508] Nucleic acids can be introduced into the cells using standardmethods which are employed for introducing a gene construct into cellswhich express the protein encoded by the gene or which express amolecule that inhibits REMODELIN expression. In some embodiments, cellsare transfected by calcium phosphate precipitation transfection, DEAEdextran transfection, electroporation, microinjection, liposome-mediatedtransfer, chemical-mediated transfer, ligand mediated transfer orrecombinant viral vector transfer.

[0509] In some embodiments, recombinant adenovirus vectors are used tointroduce DNA having a desired sequence into the cell. In someembodiments, recombinant retrovirus vectors are used to introduce DNAhaving a desired sequence into the cell. In some embodiments, standardcalcium phosphate, DEAE dextran or lipid carrier mediated transfectiontechniques are employed to incorporate a desired DNA into dividingcells. Standard antibiotic resistance selection techniques can be usedto identify and select transfected cells. In some embodiments, DNA isintroduced directly into cells by microinjection. Similarly, well knownelectroporation or particle bombardment techniques can be used tointroduce foreign DNA into cells. A second gene is usuallyco-transfected with and/or covalently linked to the nucleic acidencoding REMODELIN, or knock-out targeting vector or antisense moleculethereto. The second gene is frequently a selectableantibiotic-resistance gene. Transfected recombinant cells can beselected by growing the cells in an antibiotic that kills cells that donot take up the selectable gene. In most cases where the two genes areunlinked and co-transfected, the cells that survive the antibiotictreatment contain and express both genes.

[0510] Where an isolated REMODELIN polypeptide, an antibody thatspecifically binds with REMODELIN, a REMODELIN antisense nucleic acid,TGF-β1, TGF-β1 receptor, and/or recombinant cells of the invention areadministered to an animal either to increase or reduce the level ofREMODELIN present in the animal, one skilled in the art wouldunderstand, based upon the disclosure provided herein, that the amountof the polypeptide, nucleic acid, antibody, TGF-β1, TGF-β1 receptor, orcell to be administered to the animal can be titrated by assessing thelevel of expression of REMODELIN or the level of REMODELIN polypeptideor nucleic acid encoding REMODELIN present in the tissues of the animal.

[0511] Methods for assessing the level of REMODELIN (e.g., usinganti-REMODELIN antibodies in Western blot or other immune-based analysessuch as ELISA) and/or methods for assessing the level of REMODELINexpression in a cell and/or tissues (e.g., using Northern blot analysis,RT-PCR analysis, in situ hybridization, and the like) are disclosedherein or are well known to those skilled in the art. Such assays can beused to determine the “effective amount” of REMODELIN (whether using anisolated nucleic acid, antibody, antisense nucleic acid, ribozyme,recombinant cell, and the like) to be administered to the animal inorder to reduce or increase the level of REMODELIN to a desired level.

[0512] C. Methods of Diagnosis and Assessment of Therapies

[0513] The present invention includes methods of diagnosis certaindiseases, disorders, or conditions such as, but not limited to, negativeremodeling, arterial restenosis, adventitial fibrosis, excessive woundhealing responses, scarring, keloids, excessive bone formation, fracturehealing, ectopic ossification (malignant and benign), fibrosis in anyorgan or tissue (e.g., liver fibrosis and lung fibrosis), altered bonedensity, altered bone growth, collagen diseases, and the like, which areassociated with or mediated by abnormal expression of REMODELIN.

[0514] The invention includes a method of diagnosing tissue damage,negative remodeling, arterial restenosis, adventitial fibrosis,excessive wound healing responses, scarring, keloids, excessive boneformation, fracture healing, ectopic ossification (malignant andbenign), fibrosis in any organ or tissue (e.g., liver fibrosis and lungfibrosis), altered bone density, altered bone growth, prematurecalcification, collagen-related diseases, and the like, in a patientmammal. This is because, as demonstrated by the data disclosed herein,there is a correlation between altered expression of REMODELIN, whencompared to expression of REMODELIN in otherwise identical butundamaged, normal tissue, and tissue injury, negative remodeling,arterial restenosis, adventitial fibrosis, excessive wound healingresponses, scarring, keloids, excessive bone formation, fracturehealing, ectopic ossification (malignant and benign), fibrosis in anyorgan or tissue (e.g., liver fibrosis and lung fibrosis), altered bonedensity, altered bone growth, premature calcification, collagendiseases, and the like, such that assessing the level of REMODELINexpression is a useful diagnostic for these diseases, disorders, orconditions associated with altered expression of REMODELIN.

[0515] The method comprises obtaining a biological sample from themammal and comparing the level of REMODELIN (expression, amount,activity) in the sample with the level of REMODELIN in a sample from anormal person who is not afflicted with tissue damage, ectopicossification, and organ fibrosis. A higher level of REMODELIN in thesample from the patient compared with the level of REMODELIN in thesample obtained from a person not afflicted with tissue damage, ectopicossification, and organ fibrosis an indication that the patient isafflicted with tissue damage, ectopic ossification, and organ fibrosis.This is because, as disclosed elsewhere herein, an increased level ofREMODELIN expression is associated with tissue damage, ectopicossification, organ fibrosis, bone mineralization, skin wounding, bonedensity and/or bone growth, lack of dorsal closure, spina bifida-likephenotype, collagen-related phenotypes, and vascular injury.

[0516] In one aspect, the biological sample is selected from the groupconsisting of a lung biopsy, an aorta sample, a smooth muscle cell (SMC)sample, an endarterectomy sample, a liver biopsy, any biopsy from awound, and the like.

[0517] The invention includes a method of assessing the effectiveness ofa treatment for arterial restenosis in a mammal. The method comprisesassessing the level of REMODELIN expression, amount, and/or activity,before, during and after a specified course of treatment for arterialstenosis since arterial restenosis and/or arterial fibrosis isassociated with increased REMODELIN expression. This is because, asstated previously elsewhere and demonstrated by the data disclosedherein, REMODELIN expression, amount and/or activity is associated withor mediates decreased increased cell proliferation which is feature ofcertain disease states (e.g., negative remodeling, adventitial fibrosisand arterial restenosis). Thus, assessing the effect of a course oftreatment upon REMODELIN expression/amount/activity indicates theefficacy of the treatment such that a lower level of REMODELINexpression, amount, or activity indicates that the treatment method issuccessful.

[0518] The data disclosed herein should allow the identification andcharacterization of the REMODELIN ligand/receptor. This is useful sinceantagonism of the REMODELIN ligand, receptor, or both, should provideuseful in treatment of diseases, disorders or conditions mediated byREMODELIN ligand/receptor signaling such as, but not limited to,arterial restenosis, negative remodeling, adventitial fibrosis, fibrosisin any organ or tissue (e.g., liver, lung, among others), hypertrophicscar tissue (i.e., keloids), excessive bone formation, ectopicossification (malignant and benign), and the like.

[0519] IX. Kits

[0520] The invention includes various kits which comprise a compound,such as a nucleic acid encoding REMODELIN, an antibody that specificallybinds REMODELIN, a nucleic acid complementary to a nucleic acid encodingREMODELIN but in an antisense orientation with respect to transcription,and/or compositions of the invention, an applicator, and instructionalmaterials which describe use of the compound to perform the methods ofthe invention. Although exemplary kits are described below, the contentsof other useful kits will be apparent to the skilled artisan in light ofthe present disclosure. Each of these kits is included within theinvention.

[0521] In one aspect, the invention includes a kit for alleviating adisease mediated by altered expression of REMODELIN. The kit is usedpursuant to the methods disclosed in the invention. Briefly, the kit maybe used to contact a cell with a nucleic acid complementary to a nucleicacid encoding REMODELIN where the nucleic acid is in an antisenseorientation with respect to transcription to reduce expression ofREMODELIN, or with an antibody that specifically binds with REMODELIN,wherein the decreased expression, amount, or activity of REMODELINmediates an beneficial effect.

[0522] The kit further comprises an applicator useful for introducingthe nucleic acid or antibody protein of the invention into the cell. Theparticular applicator included in the kit will depend on, e.g., therecombinant DNA method used to introduce the nucleic acid into the celland such applicators are well-known in the art and may include, amongother things, a pipette, a syringe, a dropper, and the like. Moreover,the kit comprises an instructional material for the use of the kit.These instructions simply embody the disclosure provided herein.

[0523] The kit includes a pharmaceutically-acceptable carrier. Thecomposition is provided in an appropriate amount as set forth elsewhereherein. Further, the route of administration and the frequency ofadministration are as previously set forth elsewhere herein.

[0524] The invention is further described in detail by reference to thefollowing experimental examples. These examples are provided forpurposes of illustration only, and are not intended to be limitingunless otherwise specified. Thus, the invention should in no way beconstrued as being limited to the following examples, but rather, shouldbe construed to encompass any and all variations which become evident asa result of the teaching provided herein.

EXAMPLES

[0525] The experiments presented in this example may be summarized asfollows.

[0526] In order to identify novel factors involved in mediating arterialremodeling in response to injury, suppressive subtractive hybridizationwas performed using mRNA from normal and balloon-injured rat arteries. Anovel nucleic acid sequence was identified using this approach and afull length 1235 bp cDNA clone was isolated by screening a cDNA libraryprepared from 8 day balloon-injured rat carotid arteries and aortae.This cDNA clone comprises an open reading frame (ORF) of about 245 aminoacids having no significant homology to any known protein. This protein,referred to herein as REMODELIN, was previously termed REMODEL, and morepreviously termed AIBE for Adventitia Inducible and Bone Expressedprotein. REMODELIN comprises, inter alia, a potential transmembranedomain and five potential N-myristoylation sites which can target themolecule to the cell membrane.

[0527] In situ hybridization analysis disclosed that REMODELIN mRNAexpression is remarkably restricted to the adventitia of balloon-injuredvessels with maximal expression detected at 8 days after carotid arteryballoon denudation and with no detectable expression in normal arteries.REMODELIN expression in the adventitia was no longer detectable at 6weeks after balloon injury.

[0528] The data disclosed herein further demonstrate that during mouseembryogenesis, REMODELIN expression was prominent in developing bonestarting at about 12 days post coitus (dpc), while Northern blotanalysis demonstrated that only low levels of REMODELIN expression weredetected in brain and lung of the adult animal.

[0529] Antibodies specific for REMODELIN were generated and used tocharacterize expression of the protein by in vitro translation, invarious cell lines which endogenously express REMODELIN, injuredarteries, and cells transfected with a REMODELIN expression vector.

[0530] REMODELIN was shown to alter the expression of several cellulargenes including TGF-β1, collagen III, biglycan, osteopontin, ALP, andBMP-4. Further, REMODELIN was shown to inhibit Cbfa1-mediated activationof the osteocalcin promoter, as determined using a luciferase assay.

[0531] Injection of REMODELIN mRNA into frog embryos caused severedevelopmental abnormalities, including, but not limited to, inhibitionof FGF-induced mesoderm formation, failure of neural tissue cells tomigrate, and failure of dorsal closure, abnormal head development, andformation of a split tail.

[0532] MC3T3-E1 cells transfected with REMODELIN antisense exhibitedelongated, fibroblastic morphology, and exhibited increased cellturnover, suggesting that REMODELIN is involved in cell-matrix andcell-cell interaction(s).

[0533] Analysis of REMODELIN transgenic mice revealed severe phenotypesincluding hemorrhaging, dwarfism, skeletal abnormalities includingdecreased bone density, and severe myopathy.

[0534] The Results of the experiments presented in this example are nowdescribed.

[0535] Identification of Injury Inducible Factor

[0536] Suppressive subtractive hybridization was performed between cDNAexpressed in normal rat carotid artery/aorta and cDNA expressed in 8 dayballoon injured carotid/aorta using the PCR-Select kit from ClontechLaboratories, Inc. (Palo Alto, Calif.), to identify genes that areinvolved in the arterial remodeling response to injury. The normalvessel provided the “driver” cDNA and the injured vessel provided the“tester” cDNA.

[0537] Partial sequences of approximately 300 clones were obtained byautomated sequencing and the sequence identities were determined bysearching GenBank databases, including non-redundant and EST (expressedsequence tag) databases which are publicly available athttp://www.ncbi.nlm.nih.gov/blast/blast.cgi. Those sequences notmatching a known gene (usually corresponding murine or human ESTs) wereidentified in the database and were pursued further.

[0538] Duplicate slot blots containing the series of cDNA clones werehybridized with ³²P-dCTP labeled cDNA prepared from either normal vesselRNA or from balloon-injured vessel RNA. The clones that exhibitedincreased expression in the injured vessels were then further testedusing Northern blot technique with RNA from both normal andballoon-injured rat arteries.

[0539] The data disclosed herein demonstrate that REMODELIN expressionwas essentially not detectable in normal vessels while injured vesselsexhibited a dominant 1.2 kb transcript (FIG. 1B-1). The sequencesexhibiting detectable increased expression in the injured vesselscompared with normal vessels were further examined for expression invarious organs using Northern blot. In order to select for genes thatmight be specific for the vasculature, those clones that werepredominantly expressed in vascular tissues like lung and brain, inaddition to showing expression in the aorta or carotid artery, werepursued. The data disclosed herein demonstrate that REMODELIN showed lowlevels of expression in lung and brain (FIG. 1A-1).

[0540] Clones identified using the above-described screening approachwere then used to make ³⁵S-UTP labeled sense and antisense strand whichwere, in turn, used for in situ hybridization on normal andballoon-injured rat carotid artery sections (4, 8, 14, and 28 days afterinjury), as well as on sections from staged mouse embryos.

[0541] To determine expression in quiescent versusproliferating/migrating endothelium, en face preparations of 7 and 14day injured aortas were also used in the in situ hybridization study asdescribed in Lindner and Reidy (1993, Circ. Res. 73:589-595). In theinjured aortae, endothelial regeneration occurs from the intercostalarteries giving rise to migrating/proliferating endothelial cells at thewound edge as well as quiescent endothelium in the monolayer away fromthe wound edge.

[0542] Among the clones exhibiting modulated expression in response toinjury, REMODELIN was expressed in the adventitia of injured vessels butwas absent from normal adventitia (FIGS. 2A and 2B). Strong expressionof REMODELIN was detected at 8 days after injury with less expression at14 days (FIGS. 2B and 2C). Surprisingly, there was no appreciableREMODELIN expression detected in the media and in the developingneointima despite the fact that Smooth Muscle Cells (SMC) in vitroexpressed the 1.2 kb transcript (FIGS. 2B and 2C). No other gene isknown in the art which is specifically induced in the adventitia and isnot detectably induced in the neointima. Longer exposure or loading oflarger amounts of RNA from in vitro SMC also revealed a less abundanttranscript of about 3.5 kb (FIG. 1A-1).

[0543] At 4 weeks after balloon injury, REMODELIN expression was nearlyundetectable in the adventitia. At the time when REMODELIN is expressed,the adventitia shows rapid proliferation of myofibroblasts as well as asubsequent sharp decline in cell number that is accompanied by abundantsynthesis of collagens type I and type III (Smith et al., 1999, Cir.Res. 84:1212-1222). Interestingly, in the staged mouse embryos,REMODELIN expression was detected in the mesoderm at 11.5 days postcoitus (dpc) (FIG. 2E), which expression later became restricted to thedeveloping bone (FIG. 2F). Lower levels of REMODELIN expression werealso detected in the cortical bone of a femur from a rat pup. Duringdevelopment expression of REMODELIN was prominent in developing bonesuch as the skull (FIGS. 2F, 2G and 2H). REMODELIN continued to beexpressed in osteoblasts adjacent to mineralized bone (FIGS. 21 and 2J).

[0544] In addition, full thickness skin incisions undergoing woundhealing and remodeling revealed strong expression along the incision(FIG. 2L) while no expression was detectable in normal skin (FIG. 2K).

[0545] A full length REMODELIN clone was obtained using asize-fractionated (500 bp cut-off) cDNA library prepared using mRNAextracted from 8 day balloon-injured rat aortas and carotid arteriesusing a Lambda Zap Express system (Stratagene, La Jolla, Calif.). Afterexcision with a helper phage, the isolated cDNA clones were ligated intothe pBK-CMV vector (Stratagene, La Jolla, Calif.), which allows forconvenient expression in mammalian cells using the CMV promoter. Thislibrary is expected to contain sequences expressed in proliferating SMC,endothelial cells (EC), and fibroblasts as well as their quiescentcounterparts. In addition, sequences from inflammatory cells,predominantly macrophages, are also expected to be present in thelibrary.

[0546] The 230 bp REMODELIN clone obtained using the differential screenapproach was then used to probe the library and six clones were isolatedand sequenced. Five clones started within 50 bp upstream of a putativetranslational AUG start site at nucleotide position 116 (FIG. 4Ai). Thelongest clone contained an additional 60 bp of 5′ sequence whichcontained a potential additional in-frame AUG translational start siteat position 19 (FIG. 4Ai). The 230 bp clone was designatedREMODELIN-short (REMODELIN_(S)) and the 290 bp REMODELIN clone wasdesignated REMODELIN-long (REMODELIN_(L)).

[0547] Searching The Institute for Genomic Research (TIGR) sequencedatabase for the human homolog of REMODELIN, identified a 771 bp of 3′sequence. Using RNA from cultured human aortic SMC, 5′ RACE (rapidamplification of cDNA ends) cloning was performed to identify themissing approximately 500 bp sequence located at the 5′ end. Thesequence alignment of rat (SEQ ID NO: 2) and human REMODELIN (SEQ ID NO:4) is shown in FIG. 4. Interestingly, the human sequence did not havethe additional 5′ AUG translational start site but only contained theAUG codon at position 114. A 5′ primer located upstream of this AUGstart site and a 3′ primer were designed to verify that the AUG startsite at position 19 of the rat sequence was not a cloning artifact.Reverse transcription polymerase chain reaction (RT-PCR) analysis andsequencing was performed to confirm the presence of the AUG-19 codon.The overall identity between the human and rat REMODELIN sequence was78.3% at the nucleotide level using a blast 2 algorithm search asprovided at the web site http://www.ncbi.nlm.nih.gov/gorf/b12.html,which web site is publicly available. Further, homology between the rat(SEQ ID NO: 2) and human REMODELIN (SEQ ID NO: 4) amino acid sequencesis greater than about 97% using the blast 2 algorithm search strategydescribed previously.

[0548] Amino Acid Sequence and Domains within REMODELIN

[0549] In vitro translation was performed using a kit (Promega Corp.,Madison, Wis.) comprising rabbit reticulocyte lysate and ³⁵S-methionine.The data disclosed herein demonstrate that REMODELIN_(L) contained twoputative transcriptional start sites compatible with the Kozak rule(i.e., at positions 19 and 116) while REMODELIN_(S) had only the AUG₁₁₆site. In vitro translation revealed that the REMODELIN_(L) constructexpressed preferentially the long form but, to a lesser extent, theshort form of REMODELIN was also expressed from the REMODELIN_(L)construct (FIG. 5A). However, the REMODELIN_(S) construct expressed onlythe shorter form. The apparent molecular weights of the long and shortform were approximately 34 kDa and 30 kDa, respectively (FIG. 5A). Sincethe human sequence does not have the additional AUG codon at the 5′ end,it is most likely that in vivo translation starts at AUG₁₁₆. This ispredicted to result in a 245 amino acid (aa) rat protein while the humanhomolog has about 243 amino acids (SEQ ID NO: 4) due to a 2 amino aciddeletion in the amino terminus.

[0550] A leucine-rich hydrophobic region is located near the aminoterminus and, without wishing to be bound by any particular theory, thisregion is predicted to be a cleavable signal peptide (from about aminoacid residue number 1 to amino acid residue 32), which would result in a213 aa mature peptide having a molecular weight of about 23.1 kDa and atheoretical pI of 6.57 for the human REMODELIN. However, if the aa1-aa32peptide is not a signal sequence and does not get cleaved, it ispredicted to be a definitive transmembrane domain. The rat REMODELIN_(L)protein would result in a 277 aa protein which lacks a predicted signalsequence (SEQ ID NO: 2).

[0551] At the amino acid level, human and rat REMODELIN were 95%identical (FIG. 4B). The carboxy terminal half of the molecule was morehighly conserved with over 99% identity between the two species.Remarkably, the amino acid composition contains 4.7% cysteine, 2.4%tyrosine, and 2% tryptophan residues. A glycine-rich domain also foundin many collagens is located between amino acid residue 59 and aminoacid residue 93. A putative N-glycosylation site is located at aboutamino acid residue 188 to about amino acid residue 191, a putativeprotein kinase C (PKC) phosphorylation site at amino acid residue 146 toamino acid residue 148, and four casein kinase II (CD2) phosphorylationsites. Furthermore, there are 5 N-myristoylation sites locatedthroughout the molecule. A summary of these putative motifs is depictedin FIG. 3.

[0552] Functional Characterization of REMODELIN

[0553] Contrary to SMC in the vessel wall, cultured rat aortic SMCgrowing in DMEM supplemented with 10% bovine serum expressed REMODELINmRNA. Expression was inducible by TGF-β in 3T3 cells as well as SMC withpeak expression detected after 8 hours of stimulation. Expression levelswere still elevated after 24 hours and no induction followed stimulationof cells with FGF-2.

[0554] Since REMODELIN is also expressed in bone, expression in MC3T3cells, a bone derived cell line, was also examined. The response toTGF-β stimulation was similar to SMC and NIH3T3 cells with maximalinduction occurring after 8 hours (FIG. 1C). Stimulation with bonemorphogenetic protein-4 (BMP-4) caused a similar induction of REMODELINmRNA in MC3T3 cells as TGF-β (FIG. 1C).

[0555] Antibodies that Specifically Bind REMODELIN

[0556] The peptide corresponding to the carboxy terminal 15 amino acidsof REMODELIN was coupled to keyhole limpet hemocyanin (KLH) and used toimmunize two rabbits. One of the rabbits produced antiserum that had atiter of greater than about 1:64,000. The antiserum (used at a 1:5000dilution) detected a specific band with a molecular weight ofapproximately 30 kDa in lysates prepared from 10 day balloon-injured ratcarotid arteries while this band was absent in lysates prepared fromnormal vessels (FIG. 5C). The same specific band was also detected inlysates from cultured SMC.

[0557] In addition, SMC cultured in vitro also expressed a slightlylarger immunoreactive band of approximately 34 kDa. However,concentrated conditioned medium obtained from SMC contained nodetectable immunoreactivity, indicating that if any REMODELIN issecreted, it is at low levels beyond the level of detection. Further,the detectable bands were specific since the preimmune IgG from the samerabbit did not react with these protein bands (FIG. 5C).

[0558] Expression of GFP-tagged REMODELIN in Transfected Cells

[0559] REMODELIN cDNA was cloned into a green fluorescent protein (GFP)-and hemagglutinin (HA)-tagged expression vector to produce REMODELIN/tagpolypeptide fusion proteins. The localization of the GFP-REMODELINfusion protein was then assessed with regard to its cellularlocalization following transfection of NIH3T3 and 293 cells with theconstruct encoding the fusion protein. The data disclosed hereindemonstrate that fluorescence was distributed homogeneously throughoutthe cell with absence of GFP in the nucleus. This staining pattern iscompatible with cytosolic and/or cell membrane localization. Thetransfection efficiency obtained using the GFP-REMODELIN construct wasconsistently lower than with the GFP control vector.

[0560] Expression of myc-tagged REMODELIN in Transfected Cells

[0561] The coding region of rat REMODELIN was cloned into a mammalianexpression vector comprising a myc tag at the carboxy terminus (pcDNA3.1myc/his, Invitrogen, Carlsbad, Calif.). The sequence of the codingregion used in the construct is depicted in FIG. 10 (SEQ ID NO: 9).

[0562] Transient transfections were performed using bovine aorticendothelial cells (BAE) and NIH3T3 cells. The cell lines were analyzedfor transgene expression 24 hours and 48 hours after transfection usingWestern blotting and immunostaining using an anti-myc monoclonalantibody (Zymed, South San Francisco, Calif.).

[0563] Interestingly, the data disclosed herein demonstrate that verylittle expression remained at 48 hours post-transfection (FIG. 5F).Without wishing to be bound by any particular theory, these data suggestthat the transfected cells were lost from the culture. In comparison,the same cell line transfected in parallel with an unrelated cDNA (EP1)using the same vector exhibited significantly higher levels ofexpression at 48 hours than at 24 hours (FIG. 5F).

[0564] The possibility that overexpression of REMODELIN results in celldeath was examined further using immunohistochemistry, confocalmicroscopy and flow cytometry for cell cycle analysis. BAE cellstransiently transfected with myc-tagged REMODELIN exhibited a 15%increase in accumulation of cells in G0-G1 at 24 hours post-transfection(transfection efficiency approximately 15%). Western blotting of thesetransfectants using the anti-myc antibody demonstrated that by 48 hourspost-transfection very little immunoreactivity remained, indicating thatthe transfected cells were lost from the culture.

[0565] Confocal microscopy of REMODELIN transfected NIH3T3 cellsdemonstrated localization of the myc-tagged protein in very smallvesicles distributed throughout the cytoplasm (FIG. 5B).

[0566] Expression of REMODELIN in Various Cell Types

[0567] Vessel wall lysates were prepared from rat carotid arteriesharvested at 1, 4, 7, 14, and 28 days after balloon injury. Theselysates (30 micrograms of protein in each lane) were analyzed byimmunoblotting using an antibody raised against the carboxyterminalpeptide of REMODELIN. The highest levels of REMODELIN were seen at 4 and7 days after injury with a decline to near control levels at 28 dayspost injury (FIG. 5C).

[0568] Smooth muscle cells in vitro expressed REMODELIN mRNA while thiscell type showed very little expression in vivo. Therefore expression ofREMODELIN was examined using a variety of different cell lines usingimmunoblotting analysis (FIG. 5D). The cell lines included NIH3T3,bovine aortic (BAE), PAC-1 (a rat smooth muscle cell line), A7r5 (a ratsmooth muscle cell line), RASMC (rat aortic SMC primary culture), 293,BASMC (bovine aortic SMC), 10T1/2, human umbilical vein endothelialcells (HUVEC), A431, and human aortic SMC (HASMC). The data disclosedherein demonstrate detection of a prominent immunoreactive band with anapparent molecular weight of about 34 kDa that was present in all celllines tested. These data indicate that the antibody reacted with mouse,rat, bovine, and human homologs of REMODELIN (FIG. 5D). A less abundantprotein band having an apparent molecular weight of about 30 kDa waspresent in some cell lines and some additional larger immunoreactivebands were also detected. Without wishing to be bound by any particulartheory, the 30 kDa band may reflect differences in glycosylation orphosphorylation of REMODELIN.

[0569] Regulation of REMODELIN Expression

[0570] The regulation of REMODELIN expression by TGF-β was furtherinvestigated using MC3T3 cells, a bone derived cell line. Immunoblottingof cell lysates harvested after 24, 48, and 72 hours after stimulationwith TGF-β demonstrated increased expression levels of REMODELIN proteincompared to controls (FIG. 5E).

[0571] The data disclosed herein demonstrate that blocking signaling viathe TGF-β receptor type II by the addition of a soluble TGF-β receptortype II (Biogen, Cambridge, Mass.) to the cells, inhibited expression ofREMODELIN protein (FIG. 5E). MC3T3 cells were treated with 1 ng/ml ofTGF-β1 (TGF-β) or 100 ng/ml soluble TGF-β receptor type II (sol.TGF-βRII), the cells were harvested at various time points, and 30micrograms of protein were applied to each polyacrylamide gel lane (FIG.5E). The data disclosed herein demonstrate that TGF-β1stimulatedREMODELIN expression while inhibition of TGF-β signaling inhibitedREMODELIN expression.

[0572] Effects of REMODELIN on Gene Expression

[0573] Stable NIH3T3 and MC3T3 cell lines overexpressing REMODELIN underthe control of the CMV promoter were established. To identify geneswhose expression might be changed in response to altered levels ofREMODELIN, Northern blots from REMODELIN overexpressing and antisenseREMODELIN transfected cell lines were performed (FIG. 6). REMODELINoverexpressing NIH3T3 cells expressed considerable lower levels ofTGF-β1, collagen III, and biglycan while the mRNA levels of the samegenes were elevated in the antisense transfected cells. Most dramaticwas the >10-fold upregulation of osteopontin and ALP in the antisensetransfected cells. The dramatic effects of REMODELIN on osteopontinexpression were further analyzed using a luciferase assay. NIH3T3 cellswere cotransfected with a construct in which the osteopontin promoterdriving luciferase activity (provided by Dr. Liaw) and the REMODELINexpression construct under the control of the CMV promoter. As shown inFIG. 6, NIH3T3 cells express endogenous osteopontin. Compared to vectortransfected cells, REMODELIN decreased luciferase activity by 80% (FIG.7). As discussed herein, REMODELIN affects bone formation, and thus, itwas of interest to determine the effect of REMODELIN on Cbfa1-dependentactivity of the osteocalcin promoter. NIH3T3 and MC3T3-E1 cells werecotransfected with an osteocalcin-luciferase constrict (containing OSE2and others, provided by Dr. Karsenty), Cbfa1, and REMODELIN (or emptyvector). REMODELIN completely inhibited the Cbfa1-mediated increase inosteocalcin-driven luciferase activity (FIG. 7).

[0574] Bacterial Expression of REMODELIN

[0575] REMODELIN protein with a 6× histidine tag was expressed in E.coli, and the recombinant protein was characterized by immunoblotting.Under non-reducing conditions, bands of 74, 49, and 25 kDa reacted withanti-his tag antibody (FIG. 8, lane 1). Under reducing conditions,his-tagged recombinant REMODELIN protein runs as a single band ofapproximately 28 kDa (FIG. 8, lanes 2 and 3) which is similar in size tothe native protein detected on immunoblots (FIG. 5A). Without wishing tobe bound to any particular theory, these data suggest that REMODELIN isable to form dimers and trimers. These data indicate that REMODELIN isprobably not glycosylated and it is therefore likely that therecombinant protein has similar properties as the native REMODELIN whichmake it suitable for in vitro studies (described herein). The 28 kDaband in lane 1 probably represents reduced REMODELIN (compare with FIG.8, lanes 2 and 3).

[0576] Effects of REMODELIN Overexpression in Xenoptis laevis

[0577] The biological effects of REMODELIN overexpression on Xenopuslaevis development were studied using injection of REMODELIN mRNA intofrog embryos. For injection experiments, a dose of 5 ng of either thelong form of REMODELIN or the short form of REMODELIN mRNA was injectedinto embryos at the 2 cell stage. Controls embryos were injected with anequal volume of empty vehicle or lacZ mRNA.

[0578] Injection of both REMODELIN_(L) and REMODELIN_(S) disturbednormal embryonic development. In general, the percentage of oocytesexhibiting disturbed development was significantly higher in the oocytesinjected with the short form (close to 100%) while the long form ofREMODELIN showed fewer malformed embryos. Without wishing to be bound byany particular theory, these data may indicate that the short form ofREMODELIN is translated into protein in vivo.

[0579] In stage 17 embryos, there was a difference between lacZ-injected(FIG. 9A, left 2 embryos) and REMODELIN-injected embryos (FIG. 9A, right2 embryos) indicating inhibition of blastopore closure.

[0580] At stage 34, control-injected embryos exhibited normaldevelopment (FIG. 9B), however, REMODELIN-injected embryos displayed anumber of defects (FIG. 9C). The REMODELIN-injected embryos were smallerand were often distorted exhibiting abnormal development of the head.Due to failure of closure of the neural folds, fusion of theneurectoderm did not occur (FIG. 9D). Other malformations includeddevelopment of a split tail (FIG. 9E). Several separate injectionexperiments were performed with similar results. This phenotype isremarkably similar to that of embryos injected with mRNA fordominant-negative FGF receptor constructs (Neilson and Friesel, 1996, J.Biol. Chem. 271:250497-25057).

[0581] The effect of REMODELIN on mesoderm induction was furtherassessed in that REMODELIN or control RNA was injected at the 2 cellstage and the embryos were allowed to develop to the blastula stage, atwhich time the animal pole ectoderm (animal caps) were dissected.Uninjected animal caps incubated in the presence of 200 ng/ml of FGF-1elongated in a manner consistent with mesoderm induction whilecontrol-injected animal caps did not. Animal caps from embryos injectedwith REMODELIN and incubated in the presence of FGF-1 resembled animalcaps incubated without FGF-1. This indicates that REMODELIN was able toblock FGF-induced mesoderm formation.

[0582] Further, an important experiment assessed whether secretion ofREMODELIN is necessary for function as follows. Using PCR, a constructcomprising a deletion of the first 32 amino acids that have thepotential for being either a cleavable signal peptide or a transmembranedomain, was designed (SEQ ID NO: 9, FIG. 10). The construct was clonedinto the PCS2+ vector (American Type Culture Collection, Manassas, Va.)and RNA was injected into frog embryos at the 2 cell stage. Theresulting phenotype was similar to the one seen inREMODELIN_(S)-injected embryos both in severity as well as frequency.Without wishing to be bound by any particular theory, these resultssuggest that it is likely that REMODELIN is not a secreted protein andthat the putative N-myristoylation sites can anchor the protein in thecell membrane if required for biological function(s).

[0583] Function of REMODELIN in MC3T3 Cells

[0584] MC3T3 cells were transfected with control vector (pcDNA3.1myc/his, Invitrogen, Carlsbad, Calif.) or with full length rat REMODELINcDNA in an antisense orientation. Stably transfected clonal cell lineswere then obtained and used in cell proliferation assays.

[0585] MC3T3-E1 cells were transfected with an antisense REMODELINexpression construct and stably transfected cell lines were established.Immunoblotting of clonal cell lines demonstrated that levels ofREMODELIN protein in antisense transfectants were undetectable, ascompared with normal levels of REMODELIN protein observed in vectortransfectants (FIG. 11).

[0586] Morphology of the cells was determined using phase contrastmicroscopy for both vector transfected (FIGS. 12A through 12C) andantisense REMODELIN transfected cells. As depicted in FIGS. 12D through12I, antisense REMODELIN transfected cells exhibited a distinctlyaltered phenotype demonstrating less adhesion to the substratum andreduced cell-cell contacts. More specifically, antisense transfectedcells were elongated and fibroblastic in appearance (FIGS. 12D-I). Thisis in contrast to control vector transfected cells, which exhibited acobblestone morphology (FIGS. 12D through 12I).

[0587] The data disclosed herein also demonstrate that there wereincreased numbers of dead cells and cell debris in the antisensetransfected cells indicating, without wishing to be bound by anyparticular theory, increased cell turnover in REMODELIN antisensetransfected cells.

[0588] Increased cell turnover was determined by establishing growthcurves of the clones (FIG. 13A) and measuring [³H]-thymidineincorporation in the clones (FIG. 13B) in parallel experiments. Eventhough cell counts were similar between control and antisense REMODELINtransfected cells at all time points, [³H]-thymidine incorporation wassignificantly higher in the antisense REMODELIN transfected cells at alltime points examined (FIG. 13B). Increased cell turnover in theantisense transfected cells indicates that cell viability is reduced byshortening the cell life span. Together with the altered adhesionphenotype of the cells, the data suggests, without wishing to be boundby any particular theory, that REMODELIN is involved in cell-matrix andcell-cell interaction(s).

[0589] Expression of REMODELIN in Transgenic Mice

[0590] Transgenic mice were generated in which the coding region ofREMODELIN was under the control of the CMV promoter. Breeding of aREMODELIN transgenic female with a REMODELIN transgenic male gave riseto mouse pups that exhibited hemorrhaging in the hip and shoulderregions. The bleeding appeared to originate from the long bones (FIGS.14A and 15A), as fractures of the humerus (FIG. 15B) and femur (FIG.15C) were evident. In one instance, bleeding also occurred in smallerbones of the foot.

[0591] X-ray examination of the skeleton revealed that all transgenicmice were smaller than corresponding non-transgenic mice (FIG. 14B).This was particularly evident in the long bones.

[0592] Dissection of the dorsal skin revealed protrusion of the spinalcord similar to a phenotype seen in spina bifida disorders (FIG. 14C).

[0593] Skeletal preparations of the control and transgenic pups wereprepared using standard methods in order to further investigate theskeletal abnormalities. The skeletal preparations revealed a strikingdecrease in Alcian blue staining affecting all sites of cartilage intransgenic mice. The spinal column (FIGS. 15D and 15E) revealed avirtual absence of Alcian blue staining. Alcian blue, which binds toproteoglycans, identifies the areas of cartilage formation. The lack ofAlcian blue thus indicates a marked reduction in the proteoglycancontent of the cartilage. Alcian blue staining was also reduced in theextremities, particularly the distal phalanges. The severity of thephenotype was variable, as in some cases, the offspring from a REMODELINtransgenic/wildtype cross gave rise to severely affected mutants thatdied perinatally (FIGS. 15H through 15L). These mice were dwarfs withsevere skeletal abnormalities affecting all bones (FIGS. 15H and 15I).In addition, separation of the skin occurred which is reminiscent ofdystrophic epidermolysis bullosea (FIGS. 15H and 15L). X-ray examinationof these transgenics showed extremely reduced bone density (lack ofmineralization) and deformed bones (FIG. 15I). Masson's trichromestained sections exhibited dramatically reduced collagen matrix (blue)in many organs including skull bone (FIG. 15J) and ribs (FIG. 15K).separation of the epidermis from the dermis also appeared to result froma deficient collagen matrix (FIG. 15L). Preliminary analysis indicatedthat chondrocyte maturation appeared normal.

[0594] Mineralized bone of skeletal preparations was stained usingAlizarin Red (pink color depicted as darker gray) and cartilage wasstained using Alcian Blue (blue color depicted as lighter gray) (FIGS.16A through 16J). The data disclosed herein demonstrate that there was astriking decrease in cartilage formation affecting all sites ofcartilage generation including the extremities, particularly the distalphalanges (FIG. 16A compared with FIG. 16B). Cartilage was completelymissing from the vertebra and intervertebral joints in the REMODELINtransgenic mice (FIG. 16D) compared with normal pups (FIG. 16C), leavingthe posterior parts of the vertebrae and the intervertebral jointswithout cartilage.

[0595] The absence of cartilage in the posterior parts of the vertebrasurrounding the spinal cord is the most likely reason for the protrusionof the spinal cord leading the spina bifida phenotype. The anteriorparts of the ribs exhibited strikingly reduced cartilage formation,which was most pronounced in the more caudal ribs (FIG. 16F comparedwith FIG. 16E).

[0596] Another finding was the marked decrease in the density of themineralized bone which gave the flat bones of the skull a moretransparent appearance (compare FIG. 16H with FIG. 16G). Without wishingto be bound by any particular theory, the decreased bone density isexpected to result in weaker bones with increased tendency to fracture.Indeed, the hemorrhaging observed in the shoulder and hip regions wasfound to be the result of fractured long bones such as the humerus(FIGS. 16I and 16J) and femur.

[0597] For a better understanding of the phenotypes observed inREMODELIN transgenic mice and to clarify whether the abnormalities werethe result of ectopic expression or overexpression in cell types thathave endogenous levels of REMODELIN, the sites of transgene expressionwere characterized and compared with the expression pattern ofendogenous REMODELIN protein. Polyclonal antibodies were generated inrabbits with the recombinant protein as antigen. One of the rabbitsproduced an antibody that recognized recombinant REMODELIN and nativeREMODELIN in lung tissue lysates on immunoblots with similar sensitivityand specificity as the peptide antibody described herein. In addition,the antibody was suitable for immunohistochemistry.

[0598] A protein A-purified IgG fraction was prepared from the antiserumand the corresponding preimmune serum was used to immunostain bone, skinand muscle sections from one day old normal and REMODELIN transgenicpups. In parallel, immunostaining on sections with the transgenespecific anti-myc antibody was performed (FIGS. 17 and 18). Chondrocytesof the humerus head express endogenous REMODELIN mRNA (FIG. 17A) andendogenous REMODELIN protein was expressed abundantly by chondrocytes(FIGS. 17B and 17C). However, REMODELIN protein expression decreasedsharply in the hypertrophic cartilage zone as it reaches the osteogenicfront so that the bone matrix is essentially devoid of endogenousREMODELIN protein (FIGS. 17B and 17C). Periosteal cells and osteoblastsexpressed both REMODELIN mRNA (FIG. 17D) and protein (FIG. 17F) althoughat a lower level than chondrocytes. Expression of endogenous REMODELINin osteocytes was not detectable (FIG. 17F). The REMODELIN-myc transgeneprotein, however, was not expressed by chondrocytes (FIG. 17G, upperright corner) but high levels were synthesized by osteocytes in the bonematrix (FIG. 17H, arrowheads). Lower levels of REMODELIN-myc proteinwere also found in osteoblasts. Controls for immunostaining wereperformed with preimmune IgG, and these revealed very little nonspecificbackground (FIG. 17I). Thus, in the transition from the cartilage matrixto the bone matrix, endogenous REMODELIN and the REMODELIN-myc transgenehad inverse expression patterns (compare FIGS. 17B and 17G). Withoutwishing to be bound to any particular theory, the endogenous REMODELINexpression pattern supports the idea that REMODELIN functions as aninhibitor of mineralization, and the expression pattern of the transgeneprovides an explanation for the normal development of the cartilage inthe transgenic mice. With regard to osteoblasts, the transgenic miceprovide an in vivo overexpression model.

[0599] Immunohistochemistry performed on skin sections from normal oneday old mouse pups showed no REMODELIN protein detectable by theanti-REMODELIN IgG (FIG. 18A). It should be emphasized, however, thatskin wound repair is associated with high levels of REMODELIN mRNAexpression by dermal fibroblasts. High levels of the REMODELIN-myctransgene were expressed by keratinocytes in the epidermis as determinedby immunohistochemistry with the anti-myc antibody (FIG. 18B). Thedermis of these transgenic mice was characterized by an unusually looseconnective tissue (FIG. 18C) and hair follicles were often absent orpoorly developed. The abnormalities in the skin could explain the DEBphenotype. Skeletal muscle in normal mice revealed REMODELINimmunoreactivity in the muscle fibers (FIG. 18D) and high levels of thetransgene were also detected in skeletal muscle with the anti-mycantibody (FIG. 18G and FIG. 17G). Interestingly, the muscle fiberbundles in the transgenics were often shorter and less densely packed(FIGS. 18H and 18I). In addition, the fiber bundles often formedcircular structures that appeared hollow in the center (FIGS. 18G, 18H,and 18I). These findings indicate the presence of a severe myopathy inREMODELIN transgenic mice. With regard to skeletal muscle, thetransgenic mice provide an in vivo overexpression model. Expression ofthe transgene in skeletal muscle is consistent with data presentedherein that demonstrates activity of the CMV promoter in this tissue.

[0600] The data disclosed herein using transgenic mice indicate thatREMODELIN plays an important role in bone growth. Similar to the frogembryo injection experiments wherein REMODELIN mRNA mediated a failureof dorsal closure, the mouse transgenics also exhibit spina bifida-likedefects of the spinal column.

[0601] These findings demonstrate that altered expression of REMODELINis affecting vital mechanisms of bone formation. In particular,increased REMODELIN expression inhibits cartilage and bone formationresulting in reduced bone growth and bone mineralization which givesrise to more fragile bones. Furthermore, without wishing to be bound byany particular theory, the data disclosed herein suggest that inhibitionof REMODELIN expression can lead to the opposite phenotype withincreased cartilage formation and increased bone density and strength.The level of REMODELIN expression could thus be a predictor of boneformation, bone density and bone strength. Further, inhibiting REMODELINexpression may be useful for diseases, disorders, or conditionsassociated with decreased bone density, bone formation and bone strengthsuch as, but not limited, osteoporosis, and the like.

[0602] The data disclosed herein demonstrate that REMODELIN is expressedselectively in settings where remodeling occurs, i.e., skin incisionalwounds, bone, and the like. Without wishing to be bound by anyparticular theory, these data suggest that the role of REMODELIN is notrestricted to the vasculature but instead REMODELIN expression is berelevant to events in wound healing in general, including boneformation, bone density and bone strength.

[0603] Wound healing is characterized by the formation of granulationtissue from connective tissue surrounding the damaged area and itscomponents are inflammatory cells, fibroblasts and myofibroblasts(smooth muscle α-actin positive). As the wound closes and evolves into ascar, there is an important decrease in cellularity and a specificdisappearance of myofibroblasts (Ducy et al., 2000, Science289:1501-1504; Giachelli et al., 1993, J. Clin. Invest. 92:1686-1696).This cell loss has been shown to occur by apoptosis (Giachelli et al.,1993, J. Clin. Invest. 92:1686-1696). Failure to decrease thiscellularity may contribute to hypertrophic scarring and keloid formation(Balica et al., 1997, Circulation 95:1954-1960; Bostrom et al., 1993, J.Clin. Invest. 91:1800-1809). The response of the adventitia to ballooninjury (Bostrom et al., 1995, Amer. J. Cardiol. 75:88B-91B), and theresponse of the myocardium to infarction are very similar (Luo et al.,1997, Nature 386:78-81) with early accumulation of myofibroblasts andsubsequent loss of cells by apoptosis as disclosed elsewhere hereinresulting in an a cellular matrix-rich structure. It should beemphasized that, as demonstrated by data disclosed elsewhere herein,REMODELIN is induced in these myofibroblasts while it is not expressedin the dedifferentiated smooth muscle cells (SMC) of the neointima.While smooth muscle α-actin is down-regulated in the dedifferentiated,proliferating SMC of the neointima, it is induced in the myofibroblastsof the adventitia (Bostrom et al., 1995, Amer. J. Cardiol. 75:88B-91B),but is lost from the adventitial cells within 2 weeks after injury.

[0604] REMODELIN expression may play a role in other clinically relevantsituations of fibrosis, including liver fibrosis and pulmonary fibrosis.In liver fibrosis, apoptosis of hepatic stellate cells has beenimplicated in the fibrotic process and targeting apoptosis may be apromising strategy for antifibrotic therapies (Cales et al., 1998,Biomed. Pharmacother. 52:259-263). TGF-β has been identified as themajor factor responsible for fibrosis in the bleomycin-induced lungfibrosis model Wang et al., 1999, Thorax 54:805-812). Finally, sinceREMODELIN is expressed in developing bone it should be mentioned thatapoptosis is an integral part of endochondral ossification and bonefracture healing with chondrocytes and osteoblasts undergoing apoptosis(Einhorn et al., 1998, Clin. Orthop. S7-21; Olmedo et al., 1999, J.Orthop. Trauma 13:356-362). The prominent expression of REMODELIN indeveloping bone further suggests, without wishing to be bound by anyparticular theory, that REMODELIN is involved in regulatingcalcification since inhibition of calcification is an important event incell death (Kim, 1995, Scanning Microsc. 9:1137-1178; Kockx et al, 1998,Arterioscler. Thromb. Vasc. Biol. 18:1519-1522).

[0605] As discussed herein, mutations in collagens are responsible forOI, Bethlem myopathy, and DEB (reviewed in Spranger et al., 1994, Eur.J. Pediatr., 153:56-65). Therefore, a reasonable hypothesis is thatREMODELIN functions as a modulator of collagen matrices. Data presentedherein make it clear that it is absolutely essential that the REMODELINexpression levels be tightly regulated for normal development. Theimportance of this molecule is further demonstrated in that it is thefirst non-collagen gene to produce an OI phenotype. Based on datapresented herein, it is likely that REMODELIN is an endogenous modulatorof collagen matrices. In that capacity, the molecule could function asan endogenous regulator of (anti)fibrotic responses. The inhibition ofbone mineralization could be a consequence of a deficient collagen bonematrix that does not support mineralization. In addition, the potentialrole of REMODELIN as a signaling molecule needs to be addressed as itwas able to inhibit osteopontin promoter activity and Cbfa1-dependentactivity of the osteocalcin promoter. Furthermore, reduced expression ofcollagen was seen in cells overexpressing REMODELIN (FIG. 6). IfREMODELIN would function solely by incorporation as a component of acollagenous matrix, it should be present in matrices of healed wounds. Acompleted repair process in response to angioplasty injury of an artery,however, is associated with low REMODELIN levels (FIG. 5C, 28 day timepoint) similar to normal vessels.

[0606] In summary, the hypothesis of REMODELIN as a modulator ofcollagen matrices is based on the following findings: 1) increasedREMODELIN expression in vitro is associated with decreased TGF-βexpression as well as reduced TGF-β dependent gene expression (collagensI and III) in vivo and in vitro, 2) increased REMODELIN expression invivo results in phenotypes reminiscent of collagen mutations found inOI, DEB, and myopathies, 3) in the absence of REMODELIN, expression ofbone differentiation markers such as osteopontin and ALP aredramatically increased, and 4) the presence of a 36 aa domain withhomology to the triple helical repeat region of collagens. The proposedhypothesis for the function of REMODELIN is based on insight derivedmainly from in vivo overexpression as well as ectopic expression.

[0607] The disclosures of each and every patent, patent application, andpublication cited herein are hereby incorporated herein by reference intheir entirety.

[0608] While the invention has been disclosed with reference to specificembodiments, it is apparent that other embodiments and variations ofthis invention may be devised by others skilled in the art withoutdeparting from the true spirit and scope of the invention. The appendedclaims are intended to be construed to include all such embodiments andequivalent variations.

1 9 1 1192 DNA Rattus sp. 1 atgcggccgg ccgcagagct gggccagacg ctgagcagggccgggctctg ccgacccctt 60 tgcctcctgc tctgcgcttc gcagctaccg cacacgatgcacccccaagg ccgcgccgcc 120 tccccacagc tgctgctcgg cctcttcctt gtgctactgctgcttctgca gctgtccgcg 180 ccgtccagcg cctctgagaa tcccaaggtg aagcaaaaagcgctgatccg gcagagggaa 240 gtggtagacc tgtataatgg gatgtgccta caaggaccagcaggagttcc tggtcgcgat 300 gggagccctg gggccaatgg cattcctggc acaccgggaatcccaggtcg ggatggattc 360 aaaggagaga aaggggagtg cttaagggaa agctttgaggaatcctggac cccaaactac 420 aagcagtgtt catggagttc acttaattat ggcatagatcttgggaaaat tgcggaatgt 480 acattcacaa agatgcgatc caacagcgct cttcgagttctgttcagtgg ctcgcttcgg 540 ctcaaatgca ggaatgcttg ctgtcaacgc tggtattttacctttaatgg agctgaatgt 600 tcaggacctc ttcccattga agctatcatc tatctggaccaaggaagccc tgagttaaat 660 tcaactatta atattcatcg tacttcctcc gtggaaggactctgtgaagg gattggtgct 720 ggactggtag acgtggccat ctgggtcggc acctgttcagattaccccaa aggagacgct 780 tctactgggt ggaattctgt gtcccgcatc atcattgaagaactaccaaa ataaagcccc 840 tgaaggtttc attccctgcc tcatttactt gttaaatcaagcctctggat gggtcattta 900 aatgacattt cagaagtcac ttatgtgctc agccaaatgaaaaagcaaag ttaaatacgt 960 ttacagacca aagtgtgatc tcacacttta agatctagcattatccattt tatttcaacc 1020 aaagatggtt tcaggatttt atttctcatt gattactttttgagcctata taccggaatg 1080 ctgttatagt ctttaatatt tcctactgtt gacattttgaaacatataaa agttatgtct 1140 ttgtaagagc tgtatagaat tattttatat gttaaataaatgcttcaaac aa 1192 2 245 PRT Rattus sp. 2 Met His Pro Gln Gly Arg AlaAla Ser Pro Gln Leu Leu Leu Gly Leu 1 5 10 15 Phe Leu Val Leu Leu LeuLeu Leu Gln Leu Ser Ala Pro Ser Ser Ala 20 25 30 Ser Glu Asn Pro Lys ValLys Gln Lys Ala Leu Ile Arg Gln Arg Glu 35 40 45 Val Val Asp Leu Tyr AsnGly Met Cys Leu Gln Gly Pro Ala Gly Val 50 55 60 Pro Gly Arg Asp Gly SerPro Gly Ala Asn Gly Ile Pro Gly Thr Pro 65 70 75 80 Gly Ile Pro Gly ArgAsp Gly Phe Lys Gly Glu Lys Gly Glu Cys Leu 85 90 95 Arg Glu Ser Phe GluGlu Ser Trp Thr Pro Asn Tyr Lys Gln Cys Ser 100 105 110 Trp Ser Ser LeuAsn Tyr Gly Ile Asp Leu Gly Lys Ile Ala Glu Cys 115 120 125 Thr Phe ThrLys Met Arg Ser Asn Ser Ala Leu Arg Val Leu Phe Ser 130 135 140 Gly SerLeu Arg Leu Lys Cys Arg Asn Ala Cys Cys Gln Arg Trp Tyr 145 150 155 160Phe Thr Phe Asn Gly Ala Glu Cys Ser Gly Pro Leu Pro Ile Glu Ala 165 170175 Ile Ile Tyr Leu Asp Gln Gly Ser Pro Glu Leu Asn Ser Thr Ile Asn 180185 190 Ile His Arg Thr Ser Ser Val Glu Gly Leu Cys Glu Gly Ile Gly Ala195 200 205 Gly Leu Val Asp Val Ala Ile Trp Val Gly Thr Cys Ser Asp TyrPro 210 215 220 Lys Gly Asp Ala Ser Thr Gly Trp Asn Ser Val Ser Arg IleIle Ile 225 230 235 240 Glu Glu Leu Pro Lys 245 3 1220 DNA Homo sapiens3 acgagggcgg cctcggagcg cggcggagcc agacgctgac cacgttcctc tcctcggtct 60cctccgcctc cagctccgcg ctgcccggca gccgggagcc atgcgacccc agggccccgc 120cgcctccccg cagcggctcc gcggcctcct gctgctcctg ctgctgcagc tgcccgcgcc 180gtcgagcgcc tctgagatcc ccaaggggaa gcaaaaggcg cagctccggc agagggaggt 240ggtggacctg tataatggaa tgtgcttaca agggccagca ggagtgcctg gtcgagacgg 300gagccctggg gccaatggca ttccgggtac acctgggatc ccaggtcggg atggattcaa 360aggagaaaag ggggaatgtc tgagggaaag ctttgaggag tcctggacac ccaactacaa 420gcagtgttca tggagttcat tgaattatgg catagatctt gggaaaattg cggagtgtac 480atttacaaag atgcgttcaa atagtgctct aagagttttg ttcagtggct cacttcggct 540aaaatgcaga aatgcatgct gtcagcgttg gtatttcaca ttcaatggag ctgaatgttc 600aggacctctt cccattgaag ctataattta tttggaccaa ggaagccctg aaatgaattc 660aacaattaat attcatcgca cttcttctgt ggaaggactt tgtgaaggaa ttggtgctgg 720attagtggat gttgctatct gggttggcac ttgttcagat tacccaaaag gagatgcttc 780tactggatgg aattcagttt ctcgcatcat tattgaagaa ctaccaaaat aaatgcttta 840attttcattt gctacctctt tttttattat gccttggaat ggttcactta aatgacattt 900taaataagtt tatgtataca tctgaatgaa aagcaaagct aaatatgttt acagaccaaa 960gtgtgatttc acactgtttt taaatctagc attattcatt ttgcttcaat caaaagtggt 1020ttcaatattt ttttagttgg ttagaatact ttcttcatag tcacattctc tcaacctata 1080atttggaata ttgttgtggt cttttgtttt ttctcttagt atagcatttt taaaaaaata 1140taaaagctac caatctttgt acaatttgta aatgttaaga atttttttta tatctgttaa 1200ataaaaatta tttccaacaa 1220 4 243 PRT Homo sapiens 4 Met Arg Pro Gln GlyPro Ala Ala Ser Pro Gln Arg Leu Arg Gly Leu 1 5 10 15 Leu Leu Leu LeuLeu Leu Gln Leu Pro Ala Pro Ser Ser Ala Ser Glu 20 25 30 Ile Pro Lys GlyLys Gln Lys Ala Gln Leu Arg Gln Arg Glu Val Val 35 40 45 Asp Leu Tyr AsnGly Met Cys Leu Gln Gly Pro Ala Gly Val Pro Gly 50 55 60 Arg Asp Gly SerPro Gly Ala Asn Gly Ile Pro Gly Thr Pro Gly Ile 65 70 75 80 Pro Gly ArgAsp Gly Phe Lys Gly Glu Lys Gly Glu Cys Leu Arg Glu 85 90 95 Ser Phe GluGlu Ser Trp Thr Pro Asn Tyr Lys Gln Cys Ser Trp Ser 100 105 110 Ser LeuAsn Tyr Gly Ile Asp Leu Gly Lys Ile Ala Glu Cys Thr Phe 115 120 125 ThrLys Met Arg Ser Asn Ser Ala Leu Arg Val Leu Phe Ser Gly Ser 130 135 140Leu Arg Leu Lys Cys Arg Asn Ala Cys Cys Gln Arg Trp Tyr Phe Thr 145 150155 160 Phe Asn Gly Ala Glu Cys Ser Gly Pro Leu Pro Ile Glu Ala Ile Ile165 170 175 Tyr Leu Asp Gln Gly Ser Pro Glu Met Asn Ser Thr Ile Asn IleHis 180 185 190 Arg Thr Ser Ser Val Glu Gly Leu Cys Glu Gly Ile Gly AlaGly Leu 195 200 205 Val Asp Val Ala Ile Trp Val Gly Thr Cys Ser Asp TyrPro Lys Gly 210 215 220 Asp Ala Ser Thr Gly Trp Asn Ser Val Ser Arg IleIle Ile Glu Glu 225 230 235 240 Leu Pro Lys 5 277 PRT Rattus sp. 5 MetArg Pro Ala Ala Glu Leu Gly Gln Thr Leu Ser Arg Ala Gly Leu 1 5 10 15Cys Arg Pro Leu Cys Leu Leu Leu Cys Ala Ser Gln Leu Pro His Thr 20 25 30Met His Pro Gln Gly Arg Ala Ala Ser Pro Gln Leu Leu Leu Gly Leu 35 40 45Phe Leu Val Leu Leu Leu Leu Leu Gln Leu Ser Ala Pro Ser Ser Ala 50 55 60Ser Glu Asn Pro Lys Val Lys Gln Lys Ala Leu Ile Arg Gln Arg Glu 65 70 7580 Val Val Asp Leu Tyr Asn Gly Met Cys Leu Gln Gly Pro Ala Gly Val 85 9095 Pro Gly Arg Asp Gly Ser Pro Gly Ala Asn Gly Ile Pro Gly Thr Pro 100105 110 Gly Ile Pro Gly Arg Asp Gly Phe Lys Gly Glu Lys Gly Glu Cys Leu115 120 125 Arg Glu Ser Phe Glu Glu Ser Trp Thr Pro Asn Tyr Lys Gln CysSer 130 135 140 Trp Ser Ser Leu Asn Tyr Gly Ile Asp Leu Gly Lys Ile AlaGlu Cys 145 150 155 160 Thr Phe Thr Lys Met Arg Ser Asn Ser Ala Leu ArgVal Leu Phe Ser 165 170 175 Gly Ser Leu Arg Leu Lys Cys Arg Asn Ala CysCys Gln Arg Trp Tyr 180 185 190 Phe Thr Phe Asn Gly Ala Glu Cys Ser GlyPro Leu Pro Ile Glu Ala 195 200 205 Ile Ile Tyr Leu Asp Gln Gly Ser ProGlu Leu Asn Ser Thr Ile Asn 210 215 220 Ile His Arg Thr Ser Ser Val GluGly Leu Cys Glu Gly Ile Gly Ala 225 230 235 240 Gly Leu Val Asp Val AlaIle Trp Val Gly Thr Cys Ser Asp Tyr Pro 245 250 255 Lys Gly Asp Ala SerThr Gly Trp Asn Ser Val Ser Arg Ile Ile Ile 260 265 270 Glu Glu Leu ProLys 275 6 403 RNA Artificial Sequence Description of ArtificialSequenceREMODELIN antisense ribonucleoprobe 6 ccacccagua gaagcgucuccuuuggggua aucugaacag gugccgaccc agauggccac 60 gucuaccagu ccagcaccaaucccuucaca gaguccuucc acggaggaag uacgaugaau 120 auuaauaguu gaauuuaacucagggcuucc uugguccaga uagaugauag cuucaauggg 180 aagagguccu gaacauucagcuccauuaaa gguaaaauac cagcguugac agcaagcauu 240 ccugcauuug agccgaagcgagccacugaa cagaacucga agagcgcugu uggaucgcau 300 cuuugugaau guacauuccgcaauuuuccc aagaucuaug ccauaauuaa gugaacucca 360 ugaacacugc uuguaguuugggguccagga uuccucaaag cuu 403 7 15 PRT Artificial Sequence Descriptionof Artificial Sequencecarboxy- terminal amino acids of REMODELIN 7 GlyTrp Asn Ser Val Ser Arg Ile Ile Ile Glu Glu Leu Pro Lys 1 5 10 15 8 24PRT Artificial Sequence Description of Artificial Sequenceinsulin signalpeptide 8 Met Ala Leu Leu Val His Phe Leu Pro Leu Leu Ala Leu Leu AlaLeu 1 5 10 15 Trp Glu Pro Lys Pro Thr Gln Ala 20 9 734 DNA ArtificialSequence Description of Artificial Sequencemyc-tagged REMODELINconstruct 9 atggccccca aggccgcgcc gcctccccac agctgctgct cggcctcttccttgtgctac 60 tgctgcttct gcagctgtcc gcgccgtcca gcgcctctga gaatcccaaggtgaagcaaa 120 aagcgctgat ccggcagagg gaagtggtag acctgtataa tgggatgtgcctacaaggac 180 cagcaggagt tcctggtcgc gatgggagcc ctggggccaa tggcattcctggcacaccgg 240 gaatcccagg tcgggatgga ttcaaaggag agaaagggga gtgcttaagggaaagctttg 300 aggaatcctg gaccccaaac tacaagcagt gttcatggag ttcacttaattatggcatag 360 atcttgggaa aattgcggaa tgtacattca caaagatgcg atccaacagcgctcttcgag 420 ttctgttcag tggctcgctt cggctcaaat gcaggaatgc ttgctgtcaacgctggtatt 480 ttacctttaa tggagctgaa tgttcaggac ctcttcccat tgaagctatcatctatctgg 540 accaaggaag ccctgagtta aattcaacta ttaatattca tcgtacttcctccgtggaag 600 gactctgtga agggattggt gctggactgg tagacgtggc catctgggtcggcacctgtt 660 cagattaccc caaaggagac gcttctactg ggtggaattc tgtgtcccgcatcatcattg 720 aagaactacc aaaa 734

What is claimed is:
 1. An isolated nucleic acid encoding a mammalianREMODELIN, or a fragment thereof.
 2. The isolated nucleic acid of claim1, wherein said nucleic acid shares at least about 33% sequence identitywith a nucleic acid encoding at least one of rat REMODELIN (SEQ ID NO:1), and a human REMODELIN (SEQ ID NO: 3).
 3. An isolated nucleic acidencoding a mammalian REMODELIN, wherein the amino acid sequence of saidREMODELIN shares at least about 6% sequence identity with an amino acidsequence of at least one of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:5.
 4. An isolated polypeptide comprising a mammalian REMODELIN.
 5. Theisolated polypeptide of claim 4, wherein said mammalian REMODELINmolecule shares at least about 6% sequence identity with an amino acidsequence of at least one of SEQ ID NO: 2, SEQ ID NO: 4, and SEQ ID NO:5.
 6. The nucleic acid of claim 1, said nucleic acid further comprisinga nucleic acid encoding a tag polypeptide covalently linked thereto. 7.The nucleic acid of claim 6, wherein said tag polypeptide is selectedfrom the group consisting of a green fluorescent protein tagpolypeptide, an influenza virus hemagglutinin tag polypeptide, a myc tagpolypeptide, a glutathione-S-transferase tag polypeptide, a myc-pyruvatekinase tag polypeptide, a His6 tag polypeptide, a FLAG tag polypeptide,and a maltose binding protein tag polypeptide.
 8. The nucleic acid ofclaim 1, said nucleic acid further comprising a nucleic acid specifyinga promoter/regulatory sequence operably linked thereto.
 9. A vectorcomprising the nucleic acid of claim
 1. 10. The vector of claim 9, saidvector further comprising a nucleic acid specifying apromoter/regulatory sequence operably linked thereto.
 11. A recombinantcell comprising the isolated nucleic acid of claim
 1. 12. A recombinantcell comprising the vector of claim
 9. 13. An isolated nucleic acidcomplementary to the nucleic acid of claim 1, said complementary nucleicacid being in an antisense orientation.
 14. The isolated nucleic acid ofclaim 13, wherein said nucleic acid shares at least about 33% identitywith a nucleic acid complementary with a nucleic acid having thesequence of at least one of a rat REMODELIN molecule (SEQ ID NO: 1), anda human REMODELIN molecule (SEQ ID NO: 3).
 15. A recombinant cellcomprising the isolated nucleic acid of claim
 13. 16. An antibody thatspecifically binds with a mammalian REMODELIN molecule polypeptide, or afragment thereof.
 17. The antibody of claim 16, wherein said antibody isselected from the group consisting of a polyclonal antibody, amonoclonal antibody, a humanized antibody, a chimeric antibody, and asynthetic antibody.
 18. A composition comprising the antibody of claim16 and a pharmaceutically-acceptable carrier.
 19. A compositioncomprising the isolated nucleic acid of claim 13 and apharmaceutically-acceptable carrier.
 20. A composition comprising theisolated nucleic acid of claim 1 and a pharmaceutically-acceptablecarrier.
 21. A composition comprising the isolated polypeptide of claim4 and a pharmaceutically-acceptable carrier.
 22. A transgenic non-humanmammal comprising the isolated nucleic acid of claim
 1. 23. A method oftreating a disease mediated by abnormal expression of a REMODELINmolecule in a human, said method comprising administering to a humanpatient afflicted with a disease mediated by abnormal expression of aREMODELIN molecule a REMODELIN molecule expression-inhibiting amount ofthe composition of claim
 19. 24. The method of claim 23, wherein saiddisease is selected from the group consisting of impaired wound healing,fibrosis of an organ, ectopic ossification, and hypertrophic scarformation.
 25. A method of diagnosing arterial restenosis in a mammal,said method comprising obtaining a biological sample from said mammal,assessing the level of REMODELIN in said biological sample, andcomparing the level of REMODELIN in said biological sample with thelevel of REMODELIN in a biological sample obtained from a like mammalnot afflicted with arterial restenosis, wherein a higher level ofREMODELIN in said biological sample from said mammal compared with thelevel of REMODELIN in said biological sample from said like mammal is anindication that said mammal is afflicted with arterial restenosis,thereby diagnosing arterial restenosis in said mammal.
 26. The method ofclaim 25, wherein said biological sample is selected from the groupconsisting of a blood vessel sample, and a damaged tissue sample.
 27. Amethod of diagnosing negative remodeling in a mammal, said methodcomprising obtaining a biological sample from said mammal, assessing thelevel of REMODELIN in said biological sample, and comparing the level ofREMODELIN in said biological sample with the level of REMODELIN in abiological sample obtained from a like mammal not afflicted withnegative remodeling, wherein a higher level of REMODELIN in saidbiological sample from said mammal compared with the level of REMODELINin said biological sample from said like mammal is an indication thatsaid mammal is afflicted with negative remodeling, thereby diagnosingnegative remodeling in said mammal.
 28. A method of diagnosing fibrosisin a mammal, said method comprising obtaining a biological sample fromsaid mammal, assessing the level of REMODELIN in said biological sample,and comparing the level of REMODELIN in said biological sample with thelevel of REMODELIN in a biological sample obtained from a like mammalnot afflicted with fibrosis, wherein a higher level of REMODELIN in saidbiological sample from said mammal compared with the level of REMODELINin said biological sample from said like mammal is an indication thatsaid mammal is afflicted with fibrosis, thereby diagnosing fibrosis insaid mammal.
 29. A method of identifying a compound that affectsexpression of REMODELIN in a cell, said method comprising contacting acell with a test compound and comparing the level of REMODELINexpression in said cell with the level of REMODELIN expression in anotherwise identical cell not contacted with said test compound, whereina higher or lower level of REMODELIN expression in said cell contactedwith said test compound compared with the level of REMODELIN expressionin said otherwise identical cell not contacted with said test compoundis an indication that said test compound affects expression of REMODELINin a cell.
 30. A compound identified by the method of claim
 29. 31. Amethod of identifying a compound that reduces expression of REMODELIN ina cell, said method comprising contacting a cell with a test compoundand comparing the level of REMODELIN expression in said cell with thelevel of REMODELIN expression in an otherwise identical cell notcontacted with said test compound, wherein a lower level of REMODELINexpression in said cell contacted with said test compound compared withthe level of REMODELIN expression in said otherwise identical cell notcontacted with said test compound is an indication that said testcompound reduces expression of REMODELIN in a cell.
 32. A compoundidentified by the method of claim
 31. 33. A method of identifying acompound that affects TGF-β signaling, said method comprising contactinga cell with a test compound and comparing the level of REMODELINexpression in said cell with the level of REMODELIN expression in anotherwise identical cell not contacted with said test compound, whereina higher or lower level of REMODELIN expression in said cell contactedwith said test compound compared with the level of REMODELIN expressionin said otherwise identical cell not contacted with said test compoundis an indication that said test compound affects TGF-β signaling in acell.
 34. A kit for alleviating a disease mediated by abnormalexpression of a REMODELIN in a human, said kit comprising a REMODELINexpression-inhibiting amount of the composition of claim 19, said kitfurther comprising an applicator, and an instructional material for theuse thereof.
 35. The kit of claim 34, wherein said disease is selectedfrom the group consisting of negative remodeling, arterial restenosis,vessel injury, fibrosis.
 36. A kit for alleviating a disease mediated byabnormal expression of a REMODELIN in a human, said kit comprising aREMODELIN expression-inhibiting amount of the composition of claim 20,said kit further comprising an applicator, and an instructional materialfor the use thereof.
 37. A kit for treating a bone disease in a mammal,said kit comprising a REMODELIN expression-inhibiting amount of aninhibitor of REMODELIN expression, said kit further comprising anapplicator, and an instructional material for the use thereof.
 38. A kitfor treating a cartilage disease in a mammal, said kit comprising aREMODELIN expression-inhibiting amount of an inhibitor of REMODELINexpression, said kit further comprising an applicator, and aninstructional material for the use thereof.
 39. A kit for inhibitingtissue calcification, said kit comprising a REMODELINexpression-inhibiting amount of an inhibitor of REMODELIN expression,said kit further comprising an applicator, and an instructional materialfor the use thereof.
 40. The kit of claim 39, wherein said tissuecalcification is calcification of a transplant.
 41. The kit of claim 40,wherein said transplant is a heart valve transplant.
 42. A method ofincreasing REMODELIN expression in a mammal, said method comprisingadministering a REMODELIN expression increasing amount of TGF-β to saidmammal, thereby increasing REMODELIN expression in said mammal.
 43. Amethod of reducing REMODELIN expression in a mammal, said methodcomprising administering a REMODELIN expression reducing amount of TGF-βreceptor type II to said mammal, thereby inhibiting signaling via TGF-βreceptor type II and reducing expression of REMODELIN in said mammal.44. A method of affecting cellular gene expression in a mammal, saidmethod comprising administering a nucleic acid encoding REMODELIN tosaid mammal, thereby affecting cellular gene expression in said mammal.45. The method of claim 44, wherein said cellular gene is selected fromthe group consisting of TGF-β1, collagen IIIα1, osteopontin, biglycan,alkaline phosphatase, and bone morphogenic protein
 4. 46. The method ofclaim 45, wherein said expression of osteopontin is dependent on Cbfa1.47. A method of affecting cellular gene expression in a mammal, saidmethod comprising administering a nucleic acid antisense to a nucleicacid encoding REMODELIN to said mammal, thereby affecting cellular geneexpression in said mammal.
 48. A method of treating bone disease in amammal in need of such treatment, said method comprising administeringto a mammal afflicted with said bone disease a REMODELINexpression-inhibiting amount of an inhibitor of REMODELIN expression,thereby inhibiting REMODELIN expression and treating said bone diseasein said mammal.
 49. The method of claim 48, wherein said bone disease isosteogenesis imperfecta.
 50. A method of treating cartilage disease in amammal in need of such treatment, said method comprising administeringto a mammal afflicted with said cartilage disease a REMODELINexpression-inhibiting amount of an inhibitor of REMODELIN expression,thereby inhibiting REMODELIN expression and treating said cartilagedisease in said mammal.
 51. The method of claim 50, wherein saidcollagen disease is selected from the group consisting of osteogenesisimperfecta (OI), dystrophic epidermolysis bullosea (DEB), and Bethlemmyopathy.
 52. A method of diagnosing a bone disease in a mammal, saidmethod comprising obtaining a biological sample from said mammal,assessing the level of REMODELIN in said biological sample, andcomparing the level of REMODELIN in said biological sample with thelevel of REMODELIN in a biological sample obtained from an otherwiseidentical mammal not afflicted with bone disease, wherein a higher levelof REMODELIN in said biological sample from said mammal compared withsaid level of REMODELIN in said biological sample from said like mammalis an indication that said mammal is afflicted with bone disease,thereby diagnosing said bone disease in said mammal.
 53. The method ofclaim 52, wherein said bone disease is osteogenesis imperfecta.
 54. Amethod of diagnosing a collagen disease in a mammal, said methodcomprising obtaining a biological sample from said mammal, assessing thelevel of REMODELIN in said biological sample, and comparing the level ofREMODELIN in said biological sample with the level of REMODELIN in abiological sample obtained from an otherwise identical mammal notafflicted with a collagen disease, wherein a higher level of REMODELINin said biological sample from said mammal compared with said level ofREMODELIN in said biological sample from said like mammal is anindication that said mammal is afflicted with a collagen disease,thereby diagnosing said collagen disease in said mammal.
 55. The methodof claim 54, wherein said collagen disease is selected from the groupconsisting of osteogenesis imperfecta (OI), dystrophic epidermolysisbullosea (DEB), and Bethlem myopathy.